Compositions and methods for modulating stem cells and uses thereof

ABSTRACT

There are provided compositions and methods for modulating stem cell division decisions, in particular, division symmetry. It has been demonstrated that wnt7a acts through frizzled-7 receptor expressed on the surface of adult stem cells, e.g. satellite stem cells, to activate the planar cell polarity (PCP) pathway, thereby promoting symmetrical expansion of stem cells. The compositions and methods of the invention are useful, for example, in modulating stem cell division symmetry in vitro and in vivo, in replenishing and expanding the stem cell pool, and in promoting the formation, maintenance, repair and regeneration of tissue.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/266,428, filed Apr. 30, 2012, which is a U.S. National Entry of PCTPatent Application No. PCT/CA2010/000601, filed Apr. 27, 2010, whichclaims the benefit of priority of U.S. Patent Application Ser. No.61/172,832, filed Apr. 27, 2009, the contents of which are incorporatedherein by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates generally to compositions and methods formodulating stem cells, in particular, stem cell division symmetry, anduses thereof.

BACKGROUND OF THE INVENTION

Stem cells are undifferentiated, or immature, cells that are capable ofgiving rise to multiple specialized cell types and, ultimately, toterminally differentiated cells. Most adult stem cells arelineage-restricted and are generally referred to by their tissue origin.Unlike any other cells, stem cells are able to renew themselves suchthat a virtually endless supply of mature cell types can be generatedwhen needed over the lifetime of an organism. Due to this capacity forself-renewal, stem cells are therapeutically useful for the formation,regeneration, repair and maintenance of tissues. To ensure self-renewal,stem cells undergo two types of cell division. Symmetric division givesrise to two identical daughter cells, both endowed with stem cellproperties, and leads to expansion of the stem cell population.Asymmetric division, on the other hand, produces one stem cell and oneprogenitor cell with limited self-renewal potential. Progenitors aretransient amplifying cells that can go through several rounds of celldivision, i.e. proliferation, before terminally differentiating into amature cell. In adult organisms, stem cells and progenitor cells act asa repair system for the tissues of the body, replenish specializedcells, and maintain the normal turnover of regenerative organs, such asblood, skin or intestinal tissues.

It has recently been determined that satellite cells represent aheterogeneous population composed of stem cells and small mononuclearprogenitor cells found in mature muscle tissue (Kuang et al., 2007).Satellite cells in adult skeletal muscle are located in smalldepressions between the sarcolemma of their host myofibers and the basallamina. Satellite cells are involved in the normal growth of muscle, aswell as the regeneration of injured or diseased tissue. In undamagedmuscle, the majority of satellite cells are quiescent, meaning theyneither differentiate nor undergo cell division. Satellite cells expressa number of distinctive genetic markers, including the paired-boxtranscription factor Pax7, which plays a central regulatory role insatellite cell function and survival (Kuang et al., 2006; Seale et al.,2000). Pax7 can thus be used as a marker of satellite cells.

Upon damage, such as physical trauma or strain, repeated exercise, or indisease, satellite cells become activated, proliferate and give rise toa population of transient amplifying progenitors, which are myogenicprecursors cells (myoblasts) expressing myogenic regulatory factors(MRF), such as MyoD and Myf5. In the course of the regeneration process,myoblasts undergo multiple rounds of division before committing toterminal differentiation, fusing with the host fibers or generating newmyofibers to reconstruct damaged tissue (Charge and Rudnicki, 2004). Inseveral diseases and conditions affecting muscle, a reduction in musclemass is seen that is associated with reduced numbers of satellite cellsand a reduced ability of the satellite cells to repair, regenerate andgrow skeletal muscle. A few exemplary diseases and conditions affectingmuscle include wasting diseases, such as cachexia, muscular attenuationor atrophy, including sarcopenia, ICU-induced weakness, surgery-inducedweakness (e.g. following knee or hip replacement), and muscledegenerative diseases, such as muscular dystrophies. The process ofmuscle regeneration involves considerable remodeling of extracellularmatrix and, where extensive damage occurs, is incomplete. Fibroblastswithin the muscle deposit scar tissue, which can impair muscle function,and is a significant part of the pathology of muscular dystrophies.

Muscular dystrophies are genetic diseases characterized by progressiveweakness and degeneration of the skeletal or voluntary muscles whichcontrol movement. The muscles of the heart and some other involuntarymuscles are also affected in some forms of muscular dystrophy. In manycases, the histological picture shows variation in fiber size, musclecell necrosis and regeneration, and often proliferation of connectiveand adipose tissue. The progressive muscular dystrophies include atleast Duchenne muscular dystrophy (DMD), Becker muscular dystrophy(BMD), Emery-Dreifuss muscular dystrophy, Landouzy-Dejerine musculardystrophy, facioscapulohumeral muscular dystrophy (FSH), Limb-Girdlemuscular dystrophies, von Graefe-Fuchs muscular dystrophy,oculopharyngeal muscular dystrophy (OPMD), Myotonic dystrophy(Steinert's disease) and congenital muscular dystrophies.

Currently there is no cure for these diseases, but certain medicationsand therapies have been shown to be effective. For instance,corticosteroids have been shown to slow muscle destruction in Duchenemuscular dystrophy patients. While corticosteroids can be effective indelaying progression of the disease in many patients, long-termcorticosteroid use is undesirable due to unwanted side effects.

Researchers are also investigating the potential of certainmuscle-building medicines. One such approach is to block the proteinmyostatin, a growth factor known to play a role in the growth anddevelopment of muscle. For instance, monoclonal antibodies specific tomyostatin have been shown to improve the condition of mice with musculardystrophy, presumably by blocking the action of myostatin. Themyostatin-blocking approach presents concerns however. For instance,blocking myostatin could interfere with the satellite cells that helpreplace injured or dead muscle cells. It is believed that myostatinhelps keep satellite cells at rest until they are needed and, withoutmyostatin, the satellite cells could become depleted. In addition, ithas been proposed that myostatin blockers may be too targeted to boostmuscle growth, as there are a variety of proteins similar to myostatinthat also limit muscle growth

PCT Application No. WO 2007/059612 (Rudnicki et al.) describes a novelpopulation of Pax7+/Myf5− satellite stem cells. This group was the firstto confirm that satellite stem cells are a heterogeneous populationcontaining stem cells (Pax7+/Myf5−) and progenitor cells (Pax7+/Myf5+).Prior to this disclosure, it was unclear whether satellite cells werestem cells, committed progenitors or de-differentiated myoblasts, andwhether the niche was homogenous or heterogeneous. Using Cre/LoxPlineage-tracing, the group identified a sub-population of satellitecells which had never expressed Myf5 and functioned as a stem cellreservoir (see also Kuang et al., 2007). The group successfully isolatedthe Pax7+/Myf5− satellite stem cells, which were found to representabout 10% of the adult satellite cell pool and give rise to daughtersatellite myogenic cells (Pax7⁺/Myf5⁺) through asymmetric apical-basalcell divisions. Transplantation of both Myf5⁻ and Myf5⁺ FACS-sortedsatellite cells demonstrated that satellite stem cells are capable ofrepopulating the adult satellite cell niche as well as self-renewal(Kuang et al., 2007). It has recently been demonstrated that, duringskeletal muscle regeneration, the satellite cell population ismaintained by the stem cell subpopulation, thus allowing tissuehomeostasis and multiple rounds of regeneration during the lifespan ofan individual (Kuang et al., 2007). Knowledge of the molecular networksregulating satellite stem cell fate decisions has remained unclear.

PCT Application No. WO 2004/113513 (Rudnicki et al.) discloses methodsand compositions for modulating proliferation or lineage commitment ofan atypical population of CD45⁺Sca1⁺ stem cells, located outside thesatellite stem cell compartment, by modulating myogenic determination ofWnt proteins.

The Wnt family of genes encode over twenty cysteine-rich, secreted Wntglycoproteins that act by binding to Frizzled (Fzd) receptors on targetcells. Frizzled receptors are a family of G-protein coupled receptorproteins. Binding of different members of the Wnt-family to certainmembers of the Fzd family can initiate signaling by one of severaldistinct pathways. In the termed canonical pathway, activation of thesignaling molecule, Disheveled, leads to the inactivation of glycogensynthase kinase-3 (GSK-3β), a cytoplasmic serine-threonine kinase. TheGSK-3β target, β-catenin, is thereby stabilized and translocates to thenucleus where it activates TCF (T-cell-factor)-dependant transcriptionof specific promoters (Wodarz, 1998, Dierick, 1999). In thenon-canonical, or planar cell polarity (PCP) pathway, binding of Wnt toFzd also activates Disheveled, which in this case activates RhoA, asmall g protein. Activation of the PCP pathway does not result innuclear translocation of β-catenin.

Wnt signaling plays a key role in regulating developmental programsthrough embryonic development, and in regulating stem cell function inadult tissues (Clevers, 2006). Wnts have been demonstrated to benecessary for embryonic myogenic induction in the paraxial mesoderm(Borello et al., 2006; Chen et al., 2005; Tajbakhsh et al., 1998), aswell in the control of differentiation during muscle fiber development(Anakwe et al., 2003). Recently, the Wnt planar cell polarity (PCP)pathway has been implicated in regulating elongation of differentiatingmyocytes in the developing myotome (Gros et al., 2009). In the adult,Wnt signaling is necessary for the myogenic commitment of adultCD45+/Sca1+ stem cells in muscle tissue following acute damage(Polesskaya et al., 2003; Torrente et al., 2004). Other studies suggestthat canonical Wnt/β-catenin signaling regulates myogenicdifferentiation through activation and recruitment of reserve myoblasts.In addition, Wnt/β-catenin signaling in satellite cells within adultmuscle appears to control myogenic lineage progression by limiting Notchsignaling and thus promoting differentiation. Thus, traditionally, ithas been assumed that Wnt proteins act as stem cell growth factors,promoting the proliferation and differentiation of stem cells and/orprogenitor cells.

Stem cells, and therapies targeting stem cells, have the potential forproviding benefit in a variety of clinical settings. A limitation tomany potential therapeutic applications has been obtaining a sufficientnumber of undifferentiated stem cells, and stimulating terminaldifferentiation into mature tissue-specific cells without depleting thestem cell reservoir. Much current stem cell research focuses ondirecting the proliferation and differentiation of stem cells, inparticular, transient amplifying progenitors, to repair or regeneratedamaged tissue. In addition to concerns about stem cell depletion,another concern with stimulating proliferation and differentiation ofstem cells is abnormal or poorly-formed tissue. Accordingly, there is aneed in the art for continued research and development in the area ofstem cells and for new and improved methods and compositions formodulating stem cell function in a physiological manner.

SUMMARY OF THE INVENTION

Generally, the present invention provides compositions and methods formodulating stem cells and uses thereof. More particularly, the presentinvention provides compositions and methods for modulating stem celldivision symmetry.

Various non-limiting aspects and embodiments of the invention aredescribed below.

In one aspect, there is provided a composition for modulating thedivision symmetry of a stem cell comprising as an active agent amodulator of planar cell polarity (PCP) signaling in the stem cell.

In another aspect, there is provided a method for modulating divisionsymmetry of stem cells comprising contacting the stem cells with acomposition comprising as an active agent a modulator of planar cellpolarity (PCP) signaling in the stem cell.

In some embodiments, the active agent is an activator of PCP signalingcapable of promoting symmetrical division of the stem cell. The activeagent may, for example, comprise or be derived from a small molecule, apolynucleotide, a peptide, a polypeptide, or a combination thereof.

In some embodiments, the active agent comprises one or more of thefollowing:

(a) a peptide or polypeptide capable of binding to and/or activatingFzd7;

(b) a polynucleotide encoding a peptide or polypeptide capable ofbinding to and/or activating Fzd7;

(c) a small molecule capable of binding to and/or activating Fzd7;

(d) a polynucleotide or polypeptide capable of upregulating expressionof Fzd7 on the stem cell; or

(e) an polynucleotide or polypeptide capable of activating or inducingexpression of an effector molecule in the PCP pathway to thereby promotesymmetrical division.

In certain embodiments, the active agent comprises (a) a Wnt7apolypeptide or an active variant, fragment, analogue or derivativethereof capable of binding to and activating Fzd7, or (b) apolynucleotide encoding a Wnt7a polypeptide or an active variant,fragment, analogue or derivative thereof capable of binding to andactivating Fzd7.

In certain selected embodiments, the active agent comprises a Wnt7apolypeptide.

In other embodiments, the active agent comprises a polynucleotideencoding a Wnt7a polypeptide. The polynucleotide may, for example, bepresent in an expression vector.

In still other embodiments, the active agent modulates one or moreeffector molecules in the PCP pathway, e.g. Vangl2, α7-integrin, Prickle1 or Celsr2.

In one embodiment, the active agent is capable of inducing expression orpolarized distribution of Vangl2 in the cell membrane.

In some embodiments, the composition may comprise stem cells.

In some embodiments, the composition may comprise an inhibitor ofcanonical Wnt/β-catenin signaling in the stem cell.

In some embodiments, the composition may comprise one or more stem cellmodulators, such as, a modulator that increases the rate of stem celldivision or stem cell survival.

In some embodiments, the stem cell is an adult stem cell. In certainembodiments, the adult stem cell is a satellite stem cell.

In some embodiments, the compositions and methods of the invention areused for promoting tissue formation, regeneration, maintenance orrepair. In some embodiments, the tissue is muscle. In some embodiments,the muscle is skeletal muscle.

In one aspect, there is provided a composition for enhancing tissueformation, regeneration, maintenance or repair in a mammal comprising asan active agent (a) a Wnt7a polypeptide or an active variant, fragment,analogue or derivative thereof capable of binding to and activatingFzd7, or (b) a polynucleotide encoding a Wnt7a polypeptide or an activevariant, fragment, analogue or derivative thereof capable of binding toand activating Fzd7.

In one aspect, there is provided a method for enhancing tissueformation, regeneration, maintenance or repair in a mammal comprisingadministering to a subject in need thereof a composition comprising asan active agent (a) a Wnt7a polypeptide or an active variant, fragment,analogue or derivative thereof capable of binding to and activatingFzd7, or (b) a polynucleotide encoding a Wnt7a polypeptide or an activevariant, fragment, analogue or derivative thereof capable of binding toand activating Fzd7.

In some embodiments, the composition may be admixed with aphysiologically acceptable vehicle, carrier or diluent. In someembodiments, the composition is formulated for injection. For instance,the composition may be formulated for one or more of intravenousinjection, intramuscular injection, intracardiac injection, subcutaneousinjection, or intraperitoneal injection.

In some aspects, the compositions and methods described herein areuseful for promoting formation, maintenance, repair or regeneration ofskeletal muscle in a human subject in need thereof. For instance, thesubject in need thereof may suffer from a disease or condition affectingmuscle.

In some embodiments, the subject may have, or be suspected of having, adegenerative disease. In some embodiments, the degenerative disease is amuscular dystrophy, examples of which include, Duchenne musculardystrophy (DMD), Becker muscular dystrophy (BMD), Emery-Dreifussmuscular dystrophy, Landouzy-Dejerine muscular dystrophy,facioscapulohumeral muscular dystrophy (FSH), Limb-Girdle musculardystrophies, von Graefe-Fuchs muscular dystrophy, oculopharyngealmuscular dystrophy (OPMD), Myotonic dystrophy (Steinert's disease) andcongenital muscular dystrophies.

In some embodiments, the muscular dystrophy is Duchenne musculardystrophy (DMD).

In some embodiments, the subject suffers from muscle wasting or atrophyassociated with injury or illness.

In some embodiments, the disease or condition affecting muscle mayinclude a wasting disease (e.g. cachexia, which may be associated withan illness such as cancer or AIDS), muscular attenuation or atrophy(e.g. sarcopenia, which may be associated with aging), ICU-inducedweakness, prolonged disuse (e.g. coma, paralysis), surgery-inducedweakness (e.g. following hip or knee replacement), or a muscledegenerative disease (e.g. muscular dystrophies). This list is notexhaustive.

In another aspect, there is provided a method for modulating divisionsymmetry of stem cells in vivo or in vitro comprising contacting thestem cells with a composition comprising an effective amount of anactive agent selected from an activator or an inhibitor of planar cellpolarity (PCP) signaling in the stem cell.

In some embodiments, the method may comprise contacting stem cells withan inhibitor of canonical Wnt/β-catenin signaling in the stem cell. Suchinhibition may further promote symmetrical stem cell division.

In some embodiments, the method may comprise contacting the stem cellwith one or more stem cell modulators, for example a modulator thatincreases the rate of stem cell division or increases cell survival.

In some embodiments, the method is an in vivo method and wherein thecomposition is administered to a subject in need thereof.

In some embodiments, the method may comprise administering stem cells tothe subject. The stem cells, for example, be administered simultaneouslyor sequentially with the composition.

In some embodiments, the composition comprises stem cells. In someembodiments, the stem cells may comprise an expression vector comprisinga polynucleotide encoding Fzd7 or a modulator of PCP signaling capableof promoting symmetrical division.

In some embodiments, the method may comprise administering helper cellsto a subject. The helper cells may, for example, be administeredsimultaneously or sequentially with the composition. In someembodiments, the composition may comprise helper cells. The helper cellsmay, for example, comprise an expression vector comprising apolynucleotide encoding a Wnt7a protein or an active fragment, variant,analogue or derivative thereof capable of being secreted from the helpercell and binding to and/or activating Fzd7.

In one aspect, there is provided a method for promoting muscleformation, regeneration, maintenance or repair in a mammal comprisingadministering to the mammal a therapeutically effective amount of acomposition as herein.

In one aspect, there is provided a method for promoting muscleformation, regeneration or repair in a subject in need thereofcomprising administering to the subject a composition comprising as anactive agent (a) a Wnt7a polypeptide or an active variant, fragment,analogue or derivative thereof capable of binding to and activatingFzd7, or (b) a polynucleotide encoding a Wnt7a polypeptide or an activevariant, fragment, analogue or derivative thereof capable of binding toand activating Fzd7.

In some embodiments, a cell or tissue is transformed to overexpressWnt7a to thereby induce symmetrical stem cell division. Gene expressionmay optionally be under the control of an inducible promoter.

In another aspect, there is provided a method for preventing musclewasting, atrophy or degeneration in a subject in need thereof comprisingadministering to the subject a therapeutically effective amount of acomposition comprising (a) a Wnt7a polypeptide or an active variant,fragment, analogue or derivative thereof capable of binding to andactivating Fzd7, or (b) a polynucleotide encoding a Wnt7a polypeptide oran active variant, fragment, analogue or derivative thereof capable ofbinding to and activating Fzd7.

In another aspect, there is provided a method for expanding a populationof satellite stem cells in vivo or in vitro comprising contacting thestem cells with an effective amount of a composition comprising (a) aWnt7a polypeptide or an active variant, fragment, analogue or derivativethereof capable of binding to and activating Fzd7, or (b) apolynucleotide encoding a Wnt7a polypeptide or an active variant,fragment, analogue or derivative thereof capable of binding to andactivating Fzd7.

In some embodiments, stem cells, e.g. satellite stem cells, are expandedin vitro. In some embodiments, the in vitro expanded stem cells aresubsequently administered to a subject in need thereof.

In another aspect, there is provided a method of promoting satellitestem cell expansion comprising contacting the stem cell with Wnt7a or anactive fragment, variant, analogue or derivative thereof capable ofactivating Fzd7.

In another aspect, there is provided a use of a composition describedherein for promoting formation, maintenance, repair, or regeneration ofmuscle in a subject in need thereof.

In another aspect, there is provided a use of a composition as describedherein for the manufacture of a medicament for promoting formation,maintenance, repair, or regeneration of muscle in a subject in needthereof.

In another aspect, there is provided a use of Fxz7 as a marker ofquiescent satellite cells, wherein the marker is used in combinationwith another stem cell marker.

In another aspect, there is provided a method of identifying orisolating a satellite stem cell comprising, selecting for the markerPax7+ in combination with YFP− or Myf−.

In another aspect, there is provided a composition wherein the activeagent is an inhibitor of PCP signaling capable of inhibiting symmetricaldivision of the stem cell. The inhibitor may, for example, be a peptide,polypeptide, polynucleotide or small molecule capable of directly orindirectly inhibiting PCP signaling via inhibition of Wnt7a, Fzd7, or aneffector molecule in the PCP pathway, e.g., Vangl2, α7-integrin, Prickle1 or Celsr2.

In some embodiments, the inhibitor is a polynucleotide capable ofinhibiting expression of Wnt7a, Fzd7, or an effector molecule in the PCPpathway, e.g. siRNA or miRNA. In one embodiment, the inhibitor is Vangl2siRNA.

In another aspect, there is provided a method of screening for acompound useful in the repair or regeneration of muscle comprising:

(a) providing a population of satellite stem cells;

(b) treating the stem cells with a test compound; and

(c) determining the proportion of symmetrical to asymmetrical divisionsof the treated stem cells compared to control, wherein a increase in theproportion of symmetrical divisions compared to control indicates thatthe compound is useful in the repair or regeneration of muscle.

In another aspect, there is provided a method of screening for acompound useful in the repair or regeneration of muscle comprising:

(a) providing a population of satellite stem cells;

(b) treating the stem cells with a test compound; and

(c) determining whether the compound activates stimulates PCP signalingin the treated stems, wherein a increase in PCP signaling indicates thatthe compound is useful in the repair or regeneration of muscle.

In some embodiments, the stimulation of PCP signaling occurs viaactivation of Fzd7.

In some embodiments, the increase is an increase of at least about 10%,25%, 50%, 75% or greater.

In an aspect of the invention, there is provided a composition forpromoting symmetrical stem cell division comprising as active agent oneor more activators of the Fzd7 receptor, wherein the one or moreactivators may include, but are not limited to, one or more smallmolecules, nucleic acids, polypeptides, peptides, macromolecules or acombination thereof, that activate Fzd7 receptor in adult stem cells. Insome embodiments, the adult stem cells are satellite stem cells.

In some embodiments, a stem cell may be transformed to overexpress Fzd7.

In another aspect there is provided, a method for preventing satellitestem cell depletion comprising contacting the stem cell with (a) a Wnt7apolypeptide or an active variant, fragment, analogue or derivativethereof capable of binding to and activating Fzd7, or (b) apolynucleotide encoding a Wnt7a polypeptide or an active variant,fragment, analogue or derivative thereof capable of binding to andactivating Fzd7.

Other aspects and features of the present invention will become apparentto those ordinarily skilled in the art upon review of the followingdescription of specific embodiments of the invention in conjunction withthe accompanying Figures.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the present invention will now be described, by way ofexample only, with reference to the attached Figures, wherein:

FIG. 1: Developmental program of satellite cells in skeletal muscle.Progenitors of satellite stem cells originate in the somite as Pax3and/or Pax7 expressing progenitors. Satellite stem cells express Pax7whereas satellite myogenic cells have additionally activated Myf5transcriptional competence as revealed by expression of Myf5-lacZ andMyf5-cre knock in alleles. Following activation and entrance into thecell cycle, myogenic precursor cells express Myf5 and MyoD. Induction ofMyogenin and Mef2c together with downregulation of Myf5 then MyoD markwithdrawal from the cell cycle and entrance into the terminaldifferentiation program.

FIG. 2: The satellite cell population is heterogeneous composed ofsatellite stem cells and satellite myogenic cells. Using Myf5-Cre andROSA26-YFP Ore alleles, the present inventors found that, in vivo, about10% of sub-laminar Pax7-expressing satellite cells have never expressedMyf5. Moreover, they found that Pax7⁺/Myf5⁻ satellite cells gave rise toPax7/Myf5⁺ satellite cells through apical-basal oriented divisions thatasymmetrically generated a basal Pax7⁺/Myf5⁻ and an apical Pax7⁻/Myf5⁺cells. Prospective isolation and transplantation into muscle revealedthat whereas Pax7⁻/Myf5⁺ cells exhibited precocious differentiation,Pax7⁺/Myf5⁻ cells extensively contributed to the satellite cellreservoir throughout the injected muscle. Therefore, satellite cells area heterogeneous population composed of stem cells and committedprogenitors.

FIG. 3. Symmetric expansion of satellite stem cells results from aPCP-mediated orientation of the axis of stem cell division. Wnt7athrough binding of its receptor Fzd7 induced a polarized distribution ofVangl2 and its coreceptor Syn4 through activation of Rac/RhoA. Thissignaling leads to activation of the α7/β1-integrin receptor togetherwith its concommittent polarization as a consequence of PCP signalingthrough Fzd7 and Vangl2. The resulting upregulated and polarizedlocalization of α7/β1-integrin allows both daughter cells to remainattached to the basal lamina.

FIG. 4. Satellite Stem Cells Express the Wnt receptor Frizzled7. (A)Single myofibers isolated from Myf5-Cre/ROSA26-YFP mice. 90% of Pax7⁺cells expressed YFP, and 10% of Pax7⁺ cells were YFP⁻. Satellite cellsuniformly expressed the stem cell marker CXCR4. (B) Gated satellitecells (α7-Integrin⁺, CD34⁺, CD45⁻, CD31⁻, CD11b⁻, Sca1⁻) extracted fromresting limb skeletal muscle were separated on the basis of Myf5-Creactivated YFP fluorescence. (C) Real-time PCR analysis of sorted cellsshowing the absence of Myf5 and YFP transcripts as well as theexpression of Fzd7 transcripts in YFP⁻ sorted cells (n=3). (D) Fzd7 wasexpressed specifically in quiescent Pax7⁺/YFP⁻ satellite stem cells(left) but not in Pax7⁺/YFP⁺ satellite myogenic cells (right) in freshlyisolated Myf5-Cre/ROSA26-YFP myofibers (E) Proliferating satellite cellsand myogenic precursor cells express Fzd7. Regenerating EDL myofiberswere isolated 4 days after TA muscle injury. Both Pax7⁺/Myf5⁻ (left) andPax7⁺/Myf5⁺ (right) dividing satellite cells expressed Fzd7. Bars are 10μm. Errors bars represent SEM.

FIG. 5. Wnt7a is Highly Upregulated During Muscle Regeneration. (A)Cryosections of resting (top) and freeze-injured TA muscles analyzed at3 (middle) and 6 (bottom) days following injury. The basal lamina ofmyofibers is revealed by Laminin α2 chain staining and satellite cellnuclei were visualized by Pax7 staining. (B) Real-time PCR-arrayanalysis of regenerating TA muscle 6-days following freeze-injury,revealed upregulation of Wnt7a mRNA at the time that satellite cellsreturn to quiescence (n=3). (C) Recombinant Wnt7a protein bindsFrizzled7 at the surface of myogenic cells, and this binding isabolished after knock-down of Frizzled7. (D) Wnt3a but not Wnt7aactivates β-catenin/TCF target genes. Real-time PCR analysis of culturedmyogenic cells after stimulation with BSA (control), and recombinant Wntproteins. Only Wnt3a induced the transcription of the β-catenin/TCFtarget genes Tcf7 and Axin2 (n=5). (E) Wnt7a protein is expressed byregenerating myofibers, and not by vascular endothelial cells.Cryosections of 4-days cardiotoxin-induced regenerating (left) andresting contralateral (right) TA muscles. Sections were examined for theexpression of Myogenin (differentiating myogenic cells), CD144(endothelial cells) and Wnt7a proteins. Bars are 25 μm. Errors barsrepresent SEM.

FIG. 6. Wnt7a-Frizzled7 Signaling Drives Satellite Stem Cell Expansion.(A) First division of Pax7⁺/YFP⁻ satellite stem cells, 42 hours afterisolation of EDL single myofibers from Myf5-Cre/ROSA26-YFP mice,cultured in floating conditions. Satellite stem cells either give riseto one YFP⁻ stem cell and one YFP⁺ committed cell, via asymmetric celldivision (left), or alternatively give rise to two YFP⁻ daughter cellsby symmetric cell division (right). (B) Wnt7a but not Wnt3a stimulationmarkedly increased the proportion of symmetric cell divisions resultingin satellite stem cell expansion (n=3, *p=0.009). (C) Activatedsatellite cells on cultured myofibers at 42 h after isolation, do notexpress Fzd7 (bottom) after knock-down of Fzd7 with siRNA, as comparedto cells in non-silencing conditions (top). (D) The Wnt7a-inducedincrease in the rate of symmetric satellite stem cell divisions wasabrogated following silencing of Fzd7 on myofibers after 42 h of culture(n=3, *p<0.02). (E) The increase in symmetric satellite stem cellnumbers induced by Wnt7a was blocked by silencing of Fzd7 on myofibersafter 52 h of culture (n=3, *p<0.03). Bars are 10 μm. Errors barsrepresent SEM.

FIG. 7. PCP Components are Expressed by Myogenic Cells. (A) Quantitativereal-time PCR analysis indicated expression of PCP core componenttranscripts by YFP⁺ and YFP⁻ satellite cell-derived myoblasts (n=3). (B)Immunostaining indicated that Vangl2 is upregulated during activation ofPax7⁺ satellite cells by 24 h on cultured myofibers. (C) Wnt7a inducespolarized Vangl2 cellular localization on opposite poles of dividingPax7⁺ satellite cells on cultured myofibers. EDL myofibers were culturedin control medium or medium supplemented with Wnt7a and fixed 42 hoursafter isolation. (D) Effects of Wnt treatment on Vangl2 polarizationduring initial division. Wnt7a signaling, but not Wnt3a, inducespolarized localization of Vangl2 and Fzd7 during satellite cell division(n=3, *p=0.006). (E) Wnt7a-treated myofibers were immunolocalized forVangl2 and the membrane marker α7-Integrin. Vangl2 is polarized andco-localize to the membrane in planar-dividing satellite cells (arrows).Note the polarized and upregulated expression of α7-integrin, whichfacilitates adhesion to the basal lamina of both daughter cells. Barsare 10 μm. Errors bars represent SEM.

FIG. 8. Vangl2 is Required for Symmetric Expansion of Satellite StemCells. EDL single myofibers from Myf5-Cre/ROSA26-YFP mice were culturedin floating conditions and subjected to either non-silencing or Vangl2siRNA transfection. (A) Orientation of Pax7⁺/Syndecan4⁺ satellite cellfirst cell division at 42 hours. Divisions were scored either as planar(top) or apical-basal (bottom). Note, in myofiber culture, satellitecells translocate to the outside surface of the basal lamina andapical-basal cell divisions are directed into the media. (B) Wnt7ainduces a significant decrease in the proportion of apical-basal celldivisions after 42 h of culture supporting its function in stimulatingstem cell expansion. Knock down of Vangl2 inhibits the ability of Wnt7ato stimulate planar cell divisions (n=3, *p<0.02). (C) The Wnt7a-inducedincrease in symmetric satellite stem cell divisions was abrogatedfollowing silencing of Vangl2 on myofibers after 42 h of culture (n=3,*p<0.02). (D) Activated satellite cells on myofibers knocked-down forVangl2 after 52 h of culture do not express Vangl2 (bottom) as comparedto cells in non-silencing conditions (top). (E) Knock-down of Vangl2increased the rate of apical-basal divisions (n=5, *p=0.001). (F)Knock-down of Vangl2 decreased the proportion of Pax7⁺/YFP⁻ stem cells(n=3, *p=0.03). (G) Knock-down of Vangl2 decreased the number of cellsper fibers (n=5, *p=0.001). (H and I) Silencing of Vangl2 increased theproportion of differentiating Myogenin⁺/Pax7⁻ cells myofibers after 3days of culture (n=4, * p=10⁻⁵). (J) Silencing of Vangl2 depleted thesatellite cells pool (n=4, *p=0.001). (K) Vangl2 silencing promotesmyogenic differentiation as revealed by Real-time PCR analysis of geneexpression in satellite cell-derived myoblasts (n=4). Bars are 10 μm.Errors bars represent SEM.

FIG. 9. Ectopic Wnt7a Enhances Muscle Regeneration.

(A) Representative histology of regenerated TA muscles of 3-month oldmice, 8 days following electrotransfer-induced injury. Regeneratedmyofibers show centrally-located nuclei. Bar is 25 μm. (B)Representative cryosections of TA muscles 3 weeks followingelectroporation with CMV-Wnt7a plasmid exhibit accelerated regenerationas evidenced by increased mass, and number and caliber of fibers.Electroporation with CMV-Wnt3a resulted in malformed muscle withabnormal accumulation of matrix. The basal lamina of myofibers wasdetected by Laminin α2 chain immunostaining. Bars are 200 μm. (C)Quantification of muscle fiber caliber in TA muscles electroporated witheither saline or a Wnt7a/Wnt3a expression plasmids, as compared tocontralateral leg, 3 weeks after electroporation (n=4, *p<1.008). Wnt7aand Wnt3a have divergent effects on myofiber caliber. (D) Quantificationof muscle fiber number in TA muscles electroporated with either salineor a Wnt7a/Wnt3a expression plasmids, as compared to contralateral leg,3 weeks after electroporation (n=4, *p≤1.03). Errors bars represent SEM.

FIG. 10. Wnt7a Drives Satellite Stem Cell Expansion. (A) TA muscles of3-month old mice were electroporated with either saline or a Wnt7a/Wnt3aexpression plasmid, and dissected after 3 weeks. Sublaminar Pax7⁺satellite cells were scored on cryosections of electroporated muscles.Note the increased numbers of Pax7⁺ satellite cells followingelectroporation with CMV-Wnt7a plasmid. Bar is 25 μm. (B) The satellitecell population was increased by two-fold following electroporation ofCMV-Wnt7a plasmid (n=4, *p≤1.03), as compared to Saline− or Wnt3a−electroporated samples. (C) Satellite cells were FACS-sorted fromelectroporated Myf5-Cre/ROSA26-YFP TA muscles, 3 weeks afterelectroporation, and plated in culture for 24 hours, fixed and stainedfor Pax7 and YFP. Bar is 10 μm. (D) The proportion of Pax7⁺/YFP⁻satellite stem cells was significantly increased followingover-expression of Wnt7a in electroporated TA muscles (n=5, *p≤0.0001).(E) Wnt7a^(−/−) myofibers showed a reduced population of Pax7+ satellitecells on myofibers were isolated from EDL muscle. (n=4, *p=0.03). (F)Cryosections of freeze-injured TA muscles of 3-month old Wnt7a^(−/−)null mice and their littermate controls analyzed at 3 weeks followinginjury. No significant difference in terms of structure orcross-sectional area was observed in the regenerated muscle. Bar is 20μm. (n=3). (G) Decreased numbers of satellite cells were observed inregenerated Wnt7a^(−/−) TA muscles normalized to the number of myofibersin cross-sectional area and to the contralateral leg. (n=3, *p=0.03).Errors bars represent SEM.

FIG. 11. FACS purification of muscle satellite cells. (A) FACS profilesfor selection of live satellite cells from fore- and hindlimb skeletalmuscles. Cells were positively selected for CD34 (APC-Cy7) andα7-Integrin (APC) and negatively selected for CD11 b CD31, CD45 and Sca1(all in PE). Myf5⁺ and Myf5⁻ satellite cells were then separated on thebasis of YFP fluorescence. (B) Cytospin of freshly isolated {CD34⁺,α7-Integrin⁺, Lin⁻} satellite cells. Sorted cells express the satellitecell markers Pax7 (left) and Syndecan4 (right). (C) In vitro developmentof sorted {CD34⁺, α7-Integrin⁺, Lin⁻} satellite cells. After one week inculture, 98% of sorted cells express Pax7 (unequal levels of Pax7staining account for differences in individual myoblasts cell cycle).After 4 days in differentiation medium, sorted cells form multinucleatedmyotubes expressing Myogenin and myosin heavy chains.

FIG. 12. YFP⁻ satellite cell-derived myoblasts do not express Myf5protein. FACS-sorted YFP⁺ and YFP⁻ satellite cells were cultured in highmitogenic medium for 2 weeks. Cells were maintained at less than 10%confluence to avoid myogenic commitment of YFP⁻ cells. Cycling YFP⁺myoblasts express high levels of Myf5 protein while YFP⁻ myoblasts donot exhibit any detectable Myf5 protein expression, as revealed byimmunostaining (A) and western (B) analysis. 10T1/2 cells were used as anegative non-myogenic control (n=3).

FIG. 13. Wnt7a does not induce stabilization and nuclear localization ofβ-Catenin in activated muscle satellite cells. Single EDL myofibers werecultured in suspension for 2 days, in control medium or with mediumsupplemented with Wnt3a or Wnt7a. Myofibers were stained with anantibody recognizing the active form of fl-Catenin. Wnt3a treatmentcauses fl-Catenin stabilization and translocation into satellite cells'nucleus. Wnt7a do not activate Wnt canonical signaling in dividingsatellite cells.

FIG. 14. Wnt7a increase satellite stem cell self-renewal while notmodifying satellite cell proliferation kinetics. (A) Real-time PCRanalysis of Frizzled transcripts in cultured myogenic cells in control(non-silencing) and Fzd7-silencing conditions. Fzd7 transcription wasreduced by 80% (n=3, p=0.01, n=3). Knock-down of Fzd7 is specific anddoes not effect the expression of other Frizzled transcripts expressedin myogenic cells. (B) EDL single myofibers from Myf5-Cre/ROSA26-YFPmice were cultured in floating conditions for 52 hours. TheWnt7a-induced increase in satellite stem cell number was abrogatedfollowing silencing of Fzd7 on myofibers (n=3, *p<0.03). (C) Wnt7atreatment did not have an impact on the total number of Pax7+ cells permyofibers (n=3). Errors bars represent SEM.

FIG. 15. Efficient electroporation of plasmids in the adult TA muscle.(A) The vast majority of the TA myofibers were transfected with aCMV-LacZ expression plasmid. X-Gal staining revealing β-galactosidaseactivity in whole-mount view (left), and histology on cryosections(right). (B) Representative histology of transfected muscle 1 week afterelectroporation. Electroporation with a control plasmid (left) did notyield any significant differences in the regeneration process, ascompared to a saline electroporation (right). (C) Ectopic expression ofWnt7a by majority of the fibers 8 days after electroporation of TAmuscle with a CMV-Wnt7a expression plasmid. Immunohistochemistry ofcryosections stained with specific antibodies reactive with α2-lamininand Wnt7a.

FIG. 16. Wnt7a does not have an effect on myogenic proliferation ordifferentiation. (A) Satellite cell-derived myoblasts were grown invitro in control or Wnt supplemented growth media. Wnt7a treatment didnot alter the kinetics of committed myogenic progenitors. Wnt3atreatment resulted in a reduced cell proliferation (n=3, p=0.01). (B)Satellite cell-derived myoblasts cultured in differentiation media for24 hours were treated for 24 hours with either Wnt3a or Wnt7arecombinant proteins. Wnt treatment did not activate MyoD or Pax7transcription (n=5). Wnt3a treatment activated Axin2 transcription. (C)Satellite cell-derived myoblasts were grown to 60% confluence andshifted to control differentiation media or in differentiation mediumsupplemented with Wnt7a for 4 days. Differentiated cells wereimmunostained for myosin heavy chains. No differences in morphology orsize of the differentiated myotubes were observed between control andWnt7a-treated cultures. (D) Fusion index quantification did not show anysignificant differences between control and Wnt7a-treated cultures(n=3). (E) Myoblasts cultured in differentiation media for 24 hours weretreated for 6 hours with either Wnt3a or Wnt7a recombinant proteins.Wnt3a, but not Wnt7a, activated the transcription of Wnt-β-Catenintarget genes such as Axin2 (10-fold increase, n=3). Errors barsrepresent SEM.

FIG. 17. Electroporation of Wnt7a cDNA into the TA muscle of adult wtmice leads to an increase in satellite cell numbers. Electroporation ofWnt7a cDNA into the TA muscle resulted in an increase in the number ofsatellite cells as well as in the number of satellite stem cells (myf5negative, Pax7 positive).

FIG. 18. Number of satellite cells in mdx mice after electroporationwith a Wnt7a-containing plasmid. The TA muscle of mdx mice waselectroporated after injection of a control (lacZ) plasmid or a plasmidcontaining the coding region of Wnt7a under control of the CMV promoter.The total number of satellite cells (all Pax7 positive cells) as well assatellite stem cells (myf5 negative satellite cells) was counted. Thetotal number of satellite cells was significantly increased in mdx miceelectroporated with a Wnt7a containing plasmid (p=0.005).

FIG. 19. Electroporation of Wnt7a containing plasmid increases fiberdiameters in mdx and wt mice. Mdx mice as well as age-matched wt mice (3month old, male) were electroporated with either the Wnt7a containingplasmid or the lacZ control plasmid. Electroporation of Wnt7a cDNA leadto a significant increase in muscle fiber diameter in mdx and wt mice(p<0.001).

FIGS. 20A-B. Administration of Wnt7a recombinant protein produceseffects similar to electroporation with a Wnt7a plasmid. A. Human Wnt7aprotein was injected into the TA muscle and was found to enhance musclefibre size significantly two weeks after injection (p<0.001). B. Theobserved effects were similar to those produced by electroporation ofCMV-driven mouse Wnt7a plasmid.

FIG. 21. Mus musculus Wnt7a cDNA sequence. Mus musculus Wnt7a cDNAsequence with coding region underlined (SEQ ID NO: 1).

FIG. 22. Mus musculus Wnt7a amino acid sequence. Mus musculus Wnt7aamino acid sequence with putative mature peptide underlined (SEQ ID NO:2).

FIG. 23. Homo sapiens Wnt7a cDNA sequence. Homo sapiens Wnt7a cDNAsequence with coding region underlined (SEQ ID NO: 3).

FIG. 24. Homo sapiens Wnt7a amino acid sequence. Homo sapiens Wnt7aamino acid sequence with mature peptide underlined (SEQ ID NO: 4).

FIG. 25. Mus musculus Fzd7 cDNA sequence. Mus musculus Fzd7 cDNAsequence with coding region underlined (SEQ ID NO: 5).

FIG. 26. Mus musculus Fzd7 amino acid sequence. Mus musculus Fzd7 aminoacid sequence with putative cysteine-rich Wnt-binding domain underlined,residues defining putative Wnt-binding site bolded, and putativePDZ-domain-binding motif double underlined (SEQ ID NO: 6)

FIG. 27. Homo sapiens Fzd7 cDNA sequence. Homo sapiens Fzd7 cDNAsequence with coding region underlined (SEQ ID NO: 7).

FIG. 28. Homo sapiens Fzd7 amino acid sequence. Homo sapiens Fzd7 aminoacid sequence, with putative cysteine-rich Wnt-binding domainunderlined, residues defining putative Wnt-binding site bolded, andputative PDZ-domain-binding motif double-underlined (SEQ ID NO: 8).

FIG. 29. Mus musculus Vangl2 cDNA sequence. Mus musculus Vangl2 cDNAsequence with coding region underlined (SEQ ID NO: 9).

FIG. 30. Mus musculus Vangl2 amino acid sequence. Mus musculus Vangl2amino acid sequence with putative phosphorylation sites underlined andputative PDZ-domain-binding motif bolded (SEQ ID NO: 10).

FIG. 31. Homo sapiens Vangl2 cDNA sequence. Homo sapiens Vangl2 cDNAsequence with coding region underlined (SEQ ID NO: 11).

FIG. 32. Homo sapiens Vangl2 amino acid sequence. Homo sapiens Vangl2amino acid sequence with putative phosphorylation sites underlined andputative PDZ-domain-binding motif bolded (SEQ ID NO: 12).

DETAILED DESCRIPTION

Generally, the present invention provides compositions and methods formodulating stem cells, in particular, adult stem cells. Moreparticularly, the present invention provides compositions and methodsfor modulating stem cell division decisions.

Various uses of the compositions and methods described herein are alsoprovided, including therapeutic uses, for example, for promoting tissueformation, regeneration, repair or maintenance.

The following description details various aspects and embodiments of theinvention as contemplated by the inventors. It is understood that thescope of the invention is not limited to the exemplary embodimentsdescribed herein.

It has now been demonstrated that activation of the planar cell polarity(PCP) pathway in stem cells, e.g. adult stem cells, promotes symmetricalstem cell division. Symmetrical division gives rise to two daughtercells and results in expansion of the stem cell pool. Conversely,inhibition of PCP signaling in stem cells inhibits symmetrical division,resulting in an increase in asymmetrical (apical-basal) cell division,which does not expand the stem cell pool. Interestingly, promotion ofsymmetrical stem cell division via activation the PCP pathway had noeffect on the rate of cell division.

It has now been demonstrated that Wnt7a, acting via the Frizzled7 (Fzd7)receptor, activates of PCP signaling in adult stem cells, e.g. satellitestem cells. Satellite stem cells are adult stem cells that give rise tomuscle cells. It has further been demonstrated that inhibition ofreceptor or effector molecules in the PCP pathway, e.g. Fzd7 or Vangl2,abrogated the effects of Wnt7a. It has further been demonstratedadministration of Wnt7a polypeptide, or a polynucleotide encoding aWnt7a polypeptide, significantly increased satellite stem cell numbersin vitro and in vivo, and promoted tissue formation in vivo, leading toenhanced repair and regeneration in injured and diseased muscle tissue.

Thus, Wnt7a, Fzd7 and other components of the PCP signaling pathway arenovel targets for modulation of stem cell division decisions, andpromotion of tissue formation, regeneration, maintenance and repair.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains.

The term “stem cell”, as used herein, refers to an undifferentiated cellthat is capable of differentiating into a number of final,differentiated cell types. Different stem cells may have differentpotency. While the definitions below reflect current understanding, ourknowledge and understanding of stem cells is constantly evolving.Totipotent stem cells typically have the capacity to develop into anycell type and are usually embryonic in origin. Pluripotent stem cellsare typically cells in a stem cell line capable of differentiating intoseveral differentiated cell types. Multipotent stem cells candifferentiate into a number of cells, but only those of a closelyrelated family. Unipotent stem cells can produce only one cell type,their own, but have the property of self-renewal which distinguishesthem from non-stem cells. A muscle stem cell is an example of stem cellthat is traditionally thought to be unipotent, giving rise to musclecells only.

An “adult stem cell” is a stem cell found in a developed organism. Adultstem cells include, but are not limited to, hematopoietic stem cells,mesenchymal stem cells, neural stem cells, endothelial stem cells andmuscle stem cells.

A “satellite stem cell” is an example of an adult stem cell that givesrise to muscle cells.

The term “progenitor cell”, as used herein, refers to a cell that iscommitted to a particular cell lineage and which gives rise to cells ofthis lineage by a limited series of cell divisions. A myoblast is anexample of a progenitor cell, which is capable of differentiation toonly one type of cell, but is itself not fully mature or fullydifferentiated.

The term “symmetrical division”, as used herein in reference to stemcells, refers to a cell division that increases the number of cells ofthe same type. The term “planar division” may also be used. Symmetricalstem cell division gives rise to two daughter stem cells, therebyexpanding the stem cell pool. The term “expansion” therefore refers toan increase in the number of a cells of a particular type as a result ofsymmetrical division.

The term “asymmetrical division”, as used herein in reference to stemcells, refers to a cell division that gives rise to one daughter stemcell and one progenitor cell, with no increase in stem cell number. Theterm “apical-basal division” may also be used.

By “promoting”, “enhancing” or “increasing” symmetrical stem celldivision, it is meant that the ratio of symmetrical to asymmetrical celldivision is increased compared to normal or control, e.g. the ratio inthe absence of a particular active agent, composition or treatmentmethod. For example, the ratio of symmetrical to asymmetrical celldivision may be increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 100%, 125%, 150%, 175%, 200%, or even greater.

The term “differentiation”, as used herein, refers to a developmentalprocess whereby cells become specialized for a particular function, forexample, where cells acquire one or more morphological characteristicsand/or functions different from that of the initial cell type. The term“differentiation” includes both lineage commitment and terminaldifferentiation processes. States of undifferentiation ordifferentiation may be assessed, for example, by assessing or monitoringthe presence or absence of biomarkers using immunohistochemistry orother procedures known to a person skilled in the art.

The term “lineage commitment”, as used herein, refers to the process inwhich a stem cell becomes committed to forming a particular limitedrange of differentiated cell types. Lineage commitment arises, forexample, when a stem cell gives rise to a progenitor cell duringapical-basal division. Committed progenitor cells are often capable ofself-renewal or cell division.

The term “terminal differentiation”, as used herein, refers to the finaldifferentiation of a cell into a mature, fully differentiated cell.Usually, terminal differentiation is associated with withdrawal from thecell cycle and cessation of proliferation.

The term “Wnt” refers to a family of related genes and proteins. The Wntgenes encode over twenty cysteine-rich, secreted, Wnt proteins(glycoproteins) that act by binding to Frizzled (Fzd) receptors ontarget cells. A number of Wnt polypeptides are known in the art,including the human Wnts: Wnt 1, Wnt 2, Wnt 3, Wnt 4, Wnt 5a, Wnt 5b,Wnt 7a and Wnt 7b, and the mouse Wnts: Wnt 1, Wnt 2, Wnt 3a, Wnt 3b, Wnt4, Wnt 5a, Wnt 5b, Wnt 6, Wnt 7a, Wnt 7b, Wnt 8a, Wnt 8b, Wnt Iea, WntI0b, Wnt 11 and Wnt 12. Homologues from other species are also known andaccessible to a person skilled in the art. Members of the Wnt familydemonstrate marked evolutionary conversation and thus a high degree ofhomology is observed between species.

“Frizzled” (Fzd) receptors are a family of G-protein coupled receptorproteins to which Wnt molecules are known to bind. Sequences of variousFzd receptors are available to those skilled in the art. Fzd7 is shownherein to be expressed on satellite stem cells. Other stem cells thatexpress Fzd7 include hESC and NSC.

Binding of different members of the Wnt family to certain members of theFzd family on specific cells can initiate signaling by one of severaldistinct pathways, including canonical and non-canonical Wnt signalingpathways.

In the termed “canonical pathway”, activation of the signaling molecule,Disheveled leads to the inactivation of glycogen synthase kinase-3(GSK-38), a cytoplasmic serine-threonine kinase. The GSK-38 target,β-catenin, is thereby stabilized and translocates to the nucleus whereit activates TCF (T-cell-factor)-dependant transcription of specificpromoters. This pathway is also described as the “Wnt/β-catenin” pathwayherein. Canonical Wnt-signaling plays a well-documented role inregulating myogenic growth and differentiation.

In the termed “non-canonical” Wnt signaling pathway, also referred to asthe “planar cell polarity” (PCP) pathway, binding of Wnt to Fzd alsoactivates Disheveled (Dvl), which in this case activates RhoA, a small gprotein, triggering a cascade that is unique from the canonical pathway.For example, in contrast to the canonical pathway, activation orstimulation of the PCP pathway does not result in nuclear translocationof β-catenin.

As used herein, “effector” molecule refers to a post-receptor signalingmolecule, also referred to as a “downstream effector” molecule. Effectormolecules may include, for example, cytosolic signaling molecules ornuclear signaling molecules and transcription factors, or molecules in acell membrane, such as receptors or co-receptors. Effectors may include,for example, proteins, polynucleotides and peptides. Exemplary effectormolecules in the PCP pathway include Celsr1, Celsr2, Celsr3, Dvl1, Dvl2,Dvl3, Pk1, Pk2, Pk3, Pk4, Rac/RhoA, Vangl1, Vangl2, Syndecan 4 (Syn4)and α7-β1-integrin.

In the context of a signaling pathway, “activation” may include one ormore of, e.g. changes in phosphorylation, conformation, polarization,localization or distribution of a molecule within the cell or cellmembrane. Activation may occur directly via activation, stimulation orupregulation of an activating component of a signaling pathway, or mayoccur indirectly by inhibiting an inhibitory component. The converse isalso true where “inhibition” may occur directly or indirectly.

The term “modulator”, as used herein, refers to both “activators” and“inhibitors” of a signaling event or pathway, for example, modulators ofthe Wnt7a signaling pathway. A modulator of the Wnt7a signaling pathwaymay be a compound or molecule that stimulates or inhibits the activityor expression of a Wnt7a polypeptide, or an upstream (activator) ordownstream (effector) molecule in the Wnt7a signaling pathway, includingmodulators of the Frizzled7 (Fzd7) receptor. Candidate modulators of theWnt7a signaling pathway may stimulate or inhibit the activity of a Wnt7apolypeptide directly or indirectly. Direct modulators may act on a Wnt7apolypeptide, or a gene encoding a Wnt7a polypeptide, whereas indirectmodulators may act on one or more proteins, or genes encoding proteins,that act upstream (“activators”) or downstream (“effectors”) of a Wnt7apolypeptide in the Wnt7a signaling pathway. A modulator can act at agenetic level, for example to upregulate or downregulate the expressionof a gene encoding a Wnt7a polypeptide or an activator or effector ofWnt7a signaling, or at the protein level to interfere with the activityof a Wnt7a polypeptide or an activator or effector of Wnt7a signaling.Modulators may themselves be Wnt polypeptides, or active fragments,derivatives or variants thereof. A modulator can be, for example, apolypeptide, peptide, polynucleotide, oligonucleotide, antibody orantibody fragment, or a small molecule activator or inhibitor. Smallmolecule modulators can be organic or inorganic.

A “stem cell modulator” is a modulator that activates or inhibits afunction of a stem cell. For example, a stem cell modulator may modifystem cell division, proliferation, differentiation, or survival. Forexample, Wnt7a, is a modulator of stem cell division decisions.

The term “Wnt7a signaling pathway,” as used herein in reference to stemcells, refers to the Wnt7a-Fzd7 signaling pathway in adult stem cells,e.g. satellite stem cells, which was shown to activate PCP signaling.Wnt7a signaling was shown to induce polarized distribution of Vangl2 andα7-integrin, two known effector molecules in the PCP pathway, therebypromoting symmetrical stem cell division. Thus, the Wnt7a signalingpathway referred to herein is the PCP signaling pathway. In certainother cell types, Wnt7a may activate other Wnt signaling pathway.

Component members of the Wnt7a signaling pathway demonstrate markedevolutionary conservation, e.g. in vertebrates and mammals. Human andmouse Wnt7A proteins share about 98% sequence identity, whilecorresponding Fzd7 homologues are about 96% identical and Vangl2homologues are about 99% identical. Such high degree of homology oftenresults in cross-species activity. For instance, it has beendemonstrated herein that human Wnt7a is active in the mouse system(Example 2). Therefore, experimental findings can often be extrapolatedacross species.

The terms “protein”, “polypeptide”, and “peptide,” as used herein, referto a sequence of amino acid residues linked together by peptide bonds ormodified peptide bonds. Typically, a polypeptide is at least six aminoacids long and a peptide is at least 3 amino acids long. The polypeptideor peptide can be naturally occurring, recombinant, synthetic, or acombination of these. The polypeptide or peptide can be a fragment of anaturally occurring protein or polypeptide. The terms polypeptide andpeptide also encompass analogues, derivatives and peptidomimeticcompounds. Such compounds are well known in the art and may havesignificant advantages over naturally occurring peptides, including, forexample, greater chemical stability, increased resistance to proteolyticdegradation, enhanced pharmacological properties (such as, half-life,absorption, potency and efficacy), altered specificity (for example, abroad-spectrum of biological activities) or reduced antigenicity.

Specific proteins or polypeptides (e.g. Wnt7a, Fzd7 or Vangl2, etc.)referred to herein encompass proteins and polypeptides having amino acidsequences corresponding to naturally occurring sequences, as well asvariant or homologous polypeptide sequences, fragments and derivativeshaving an activity at least substantially identical to a wild-typeprotein. Likewise, specific genes (e.g. Wnt7a, Fzd7 or Vangl2, etc.)encompass nucleic acid sequences or partial sequences encoding proteinshaving a polypeptide sequence corresponding to naturally occurringsequences as well as variant or homologous polypeptide sequences,fragments, analogies and derivatives having an activity at leastsubstantially identical to a wild-type protein. Polypeptides, includingvariants, fragments, analogues and derivatives thereof, having anincreased activity compared to wild-type polypeptides are alsocontemplated.

A functional “activity”, as used herein in reference to a polypeptide orgene or portion thereof, refers to a polypeptide, gene or portionthereof that displays one or more activities associated with anaturally-occurring protein or gene. Functional activity in regard to apolypeptide or portion thereof may include, for example, the ability tospecifically bind to and/or activate a receptor or ligand for thepolypeptide.

“Naturally occurring”, as used herein in reference to an object,indicates that the object can be found in nature. For example, anaturally occurring polypeptide or polynucleotide sequence would be onethat is present in an organism, and can be isolated from the organism,and which has not been intentionally modified by man in the laboratory.The term “wild-type” is often used interchangeably with naturallyoccurring.

In the context of the present invention, a polypeptide, or fragment,variant, analogue or derivative thereof, is considered to have at leastsubstantially the same activity as the wild-type protein when itexhibits about 50% of the activity of the wild-type protein, preferablyat least 60%, 75%, or 80% of the activity of the wild-type protein. Inpreferred embodiments, the polypeptide, variant, fragment analogue orderivative exhibits at least about 85% of the activity of the wild-typeprotein, e.g. 88%, 90%, 95%, 99%, 100%. In certain embodiments, anactivity greater than wild-type activity may be achieved. Activity of aWnt7a polypeptide, variant, fragment, analogue or derivative can, forexample, be determined by measuring its ability to promote symmetricalstem cell expansion and comparing to a wild-type protein. Methods ofmeasuring and characterizing stem cell division are known in the art.

A “fragment” of a polypeptide includes, but is not limited to, an aminoacid sequence wherein one or more amino acids are deleted in comparisonto the wild-type sequence or another reference sequence. For example,but not to be considered limiting, a fragment exists when one or moreamino acids from the amino terminal, carboxy terminal or both areremoved. Further, one or more amino acids internal to the polypeptidemay be deleted. Active fragments are fragments that retain functionalcharacteristics, e.g. of the native sequence or other referencesequence. Typically, active fragments are fragments that retainsubstantially the same activity as the wild-type protein. A fragmentmay, for example, contain a functionally important domain, such as adomain that is important for receptor or ligand binding.

A “variant” polypeptide or variant fragment is one in which one or moreamino acid residues have been deleted, added, or substituted for thosethat appear in the amino acid sequence of a wild-type sequence oranother reference sequence. In the context of the present invention, avariant preferably retains substantially the same activity as thewild-type sequence or other reference sequence, or has better activitythan the wild type protein.

A variant may contain one or more amino acid substitutions, which may be“conservative” or “non-conservative” substitutions. As is known in theart, the twenty naturally occurring amino acids can be grouped accordingto the physicochemical properties of their side chains. Suitablegroupings include alanine, valine, leucine, isoleucine, proline,methionine, phenylalanine and tryptophan (hydrophobic side chains);glycine, serine, threonine, cysteine, tyrosine, asparagine, andglutamine (polar, uncharged side chains); aspartic acid and glutamicacid (acidic side chains) and lysine, arginine and histidine (basic sidechains). Another grouping of amino acids is phenylalanine, tryptophan,and tyrosine (aromatic side chains). A conservative substitutioninvolves the substitution of an amino acid with another amino acid fromthe same group, while a non-conservative substitution involves thesubstitution of an amino acid with another amino acid from a differentgroup.

Typically, variant amino acid sequences comprise greater than about 70%,more preferably greater than about 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% identity to the wild-type or referencesequence. The degree of identity may also be represented by a rangedefined by any two of the values listed above or any value thereinbetween. Variants include “mutants”, in which the reference sequence isthe wild-type sequence.

A “derivative” is a peptide or polynucleotide containing additionalchemical or biochemical moieties not normally a part of a naturallyoccurring molecule. Peptide derivatives include peptides in which one ormore amino acid side chain and/or the amino-terminus and/or thecarboxy-terminus has been derivatized with a suitable chemicalsubstituent group, as well as cyclic peptides, dual peptides, multimersof the peptides, peptides fused to other proteins or carriersglycosylated peptides, phosphorylated peptides, peptides conjugated tolipophilic moieties (for example, caproyl, lauryl, stearoyl moieties)and peptides conjugated to an antibody or other biological ligand.Examples of chemical substituent groups that may be used to derivatize apeptide include, but are not limited to, alkyl, cycloalkyl and arylgroups; acyl groups, including alkanoyl and aroyl groups; esters;amides; halogens; hydroxyls; carbamyls, and the like. The substituentgroup may also be a blocking group such as Fmoc (fluorenylmethyl-O—CO—),carbobenzoxy(benzyl-CO—), monomethoxysuccinyl naphthyl-NH—CO—,acetylamino-caproyl and adamantyl-NH—CO—. Other derivatives includeC-terminal hydroxymethyl derivatives, O-modified derivatives (forexample, C-terminal hydroxymethyl benzyl ether) and N-terminallymodified derivatives including substituted amides such as alkylamidesand hydrazides.

An “analogue” is a polypeptide or peptide comprising one or morenon-naturally occurring amino acids. As is known in the art,substitution of all D-amino acids for all L-amino acids within a peptidecan result in an “inverse” peptide, or in a “retro-inverso” peptide (seeGoodman et al. “Perspectives in Peptide Chemistry” pp. 283-294 (1981);U.S. Pat. No. 4,544,752), both of which are considered to be analoguesin the context of the present invention. An “inverse” peptide is one inwhich all L-amino acids of a sequence have been replaced with D-aminoacids, and a “retro-inverso” peptide is one in which the sequence of theamino acids has been reversed (“retro”) and all L-amino acids have beenreplaced with D-amino acids.

“Peptidomimetics” are compounds that are structurally similar topeptides and contain chemical moieties that mimic the function of thepolypeptide or peptide of the invention. For example, if a polypeptidecontains two charged chemical moieties having functional activity, amimetic places two charged chemical moieties in a spatial orientationand constrained structure so that the charged chemical function ismaintained in three-dimensional space. The term peptidomimetic thus isintended to include isosteres. The term “isostere” refers to a chemicalstructure that can be substituted for a polypeptide or peptide becausethe steric conformation of the chemical structure is similar to that ofthe peptide or polypeptide, for example, the structure fits a bindingsite specific for the polypeptide or peptide.

One skilled in the art will appreciate that not all amino acids in apeptide or polypeptide need be modified. Similarly not all amino acidsneed be modified in the same way. Peptide derivatives, analogues andpeptidomimetics of the present invention include chimeric moleculeswhich contain two or more chemically distinct regions, each regioncomprising at least one amino acid or modified version thereof.

A “Wnt7a polypeptide,” as used herein, encompasses a Wnt 7a proteinhaving a polypeptide sequence corresponding to a wild-type Wnt7asequence, or having a sequence that is at least about as 70%, morepreferably about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or about 100%, identical to a naturally occurring Wnt7a sequence.Identity may be assessed over at least about 50, 100, 200, 300, or morecontiguous amino acids, or may be assessed over the full length of thesequence. Methods for determining % identity or % homology are known inthe art and any suitable method may be employed for this purpose. Wnt7apolypeptides also include variants, fragments, analogues and derivativeshaving an activity substantially identical to a wild-type Wnt7apolypeptide, e.g. binding to Fzd7. U.S. Pat. No. 6,297,030 describesWnt7a polypeptides and polynucleotides and methods for producing suchpolypeptides by recombinant techniques. Exemplary Wnt7A polypeptidesinclude polypeptides comprising the amino acid sequence shown in SEQ IDNO: 2 (mouse) or SEQ ID NO: 4 (human), as well as active fragments,variants or derivatives thereof.

The polypeptides of the present invention can be prepared by methodsknown in the art, such as purification from cell extracts or the use ofrecombinant techniques. Polypeptides as described herein will preferablyinvolve purified or isolated polypeptide preparations. In certainembodiments, purification of the polypeptide may utilize recombinantexpression methods well known in the art, and may involve theincorporation of an affinity tag into the expression construct to allowfor affinity purification of the target polypeptide.

Shorter sequences can also be chemically synthesized by methods known inthe art including, but not limited to, exclusive solid phase synthesis,partial solid phase synthesis, fragment condensation or classicalsolution synthesis (Merrifeld (1963) Am. Chem. Soc. 85:2149; Merrifeld(1986) Science 232:341). The polypeptides of the present invention canbe purified using standard techniques such as chromatography (e.g. ionexchange, affinity, and sizing column chromatography or high performanceliquid chromatography), centrifugation, differential solubility, or byother techniques familiar to a worker skilled in the art. Thepolypeptides can also be produced by recombinant techniques. Typicallythis involves transformation (including transfection, transduction, orinfection) of a suitable host cell with an expression vector comprisinga polynucleotide encoding the protein or polypeptide. The nucleic acidsequences for human and mouse wnt7a gene and various other components ofthe PCP signaling pathway are known in the art (see, for example,GenBank Accession Nos O00755, P24383, NP_004616, G36470, PF6706, P28047,H36470, NM_004625, M89801) and may be used as a basis for thepolynucleotides of the invention.

The polypeptides and peptides of the present invention can also beproduced as fusion proteins. One use of such fusion proteins is toimprove the purification or detection of the polypeptide or peptide. Forexample, a polypeptide or peptide can be fused to an immunoglobulin Fcdomain and the resultant fusion protein can be readily purified using aprotein A column. Other examples of fusion proteins include polypeptidesor peptides fused to histidine tags (allowing for purification on Nie+resin columns), to glutathione-S-transferase (allowing purification onglutathione columns) or to biotin (allowing purification on streptavidincolumns or with streptavidin labelled—19 magnetic beads). Once thefusion protein has been purified, the tag may be removed bysite-specific cleavage using chemical or enzymatic methods known in theart.

The term “gene” encompasses the coding regions of a structural gene andincludes sequences located adjacent to the coding region on both the 5′and 3′ ends for a distance of about 1 kb on either end such that thegene corresponds to the length of the full-length mRNA. The sequenceswhich are located 5′ of the coding region and which are present on themRNA are referred to as 5′ non-translated sequences. The sequences whichare located 3′ or downstream of the coding region and which are presenton the mRNA are referred to as 3′ non-translated sequences. The term“gene” encompasses both cDNA and genomic forms of a gene. A genomic formor clone of a gene contains the coding region termed “exon” or“expressed regions” or “expressed sequences” interrupted with non-codingsequences termed “introns” or “intervening regions” or “interveningsequences.” Introns are segments of a gene that are transcribed intonuclear RNA (hnRNA); introns may contain regulatory elements such asenhancers. Introns are removed or “spliced out” from the nuclear orprimary transcript; introns therefore are absent in the messenger RNA(mRNA) transcript. The mRNA functions during translation to specify thesequence or order of amino acids in a nascent polypeptide. In additionto containing introns, genomic forms of a gene may also includesequences located on both the 5′ and 3′ end of the sequences that arepresent on the RNA transcript. These sequences are referred to as“flanking” sequences or regions (these flanking sequences are located 5′or 3′ to the non-translated sequences present on the mRNA transcript).The 5′ flanking region may contain regulatory sequences such aspromoters and enhancers that control or influence the transcription ofthe gene. The 3′ flanking region may contain sequences that direct thetermination of transcription, posttranscriptional cleavage andpolyadenylation.

As used herein, the term “polynucleotide sequence” or “nucleic acidsequence,” refers to any nucleotide sequence (e.g., RNA or DNA), themanipulation of which may be deemed desirable for any reason (e.g.,modulate cell function, treat disease, etc.), by one of ordinary skillin the art. Such nucleotide sequences include, but are not limited to,coding sequences of genes (e.g., reporter genes, selection marker genes,oncogenes, disease resistance genes, growth factors, etc.), andnon-coding regulatory sequences which may not encode an mRNA (e.g.,promoter sequence, polyadenylation sequence, termination sequence,enhancer sequence, etc.).

The term “oligonucleotide” refers to a molecule comprised of two or moredeoxyribonucleotides or ribonucleotides, preferably more than three, andusually more than ten. The exact size will depend on many factors,which, in turn, depend on the ultimate function or use of theoligonucleotide. The oligonucleotide may be generated in any manner,including chemical synthesis, DNA replication, reverse transcription, ora combination thereof.

By the terms “regulatory sequence”, “regulatory region”, “regulatoryelement” it is meant a portion of nucleic acid typically, but notalways, upstream of the protein or polypeptide coding region of anucleotide sequence, which may be comprised of either DNA or RNA, orboth DNA and RNA. When a regulatory region is active, and in operativeassociation with a nucleotide sequence of interest, this may result inexpression of the nucleotide sequence of interest. A regulatory elementmay be capable of mediating organ specificity, or controllingdevelopmental or temporal nucleotide sequence activation. A “regulatoryregion” includes promoter elements, core promoter elements exhibiting abasal promoter activity, elements that are inducible in response to astimulus, elements that mediate promoter activity such as negativeregulatory elements or transcriptional enhancers. “Regulatory region”,as used herein, also includes elements that are active followingtranscription, for example, regulatory elements that modulate nucleotidesequence expression such as translational and transcriptional enhancers,translational and transcriptional repressors, upstream activatingsequences, and mRNA instability determinants. Several of these latterelements may be located proximal to the coding region.

The terms “complementary” and “complementarity” refer to polynucleotides(i.e., a sequence of nucleotides) related by the base-pairing rules. Forexample, the sequence “A-G-T-” is complementary to the sequence “T-C-A.”Complementarity may be “partial,” in which only some of the nucleicacids' bases are matched according to the base pairing rules. Or, theremay be “complete” or “total” complementarity between the nucleic acids.The degree of complementarity between nucleic acid strands hassignificant effects on the efficiency and strength of hybridizationbetween nucleic acid strands. This is of particular importance inamplification reactions, as well as detection methods that depend uponbinding between nucleic acids.

The term “recombinant” when made in reference to a nucleic acid moleculerefers to a nucleic acid molecule that is comprised of segments ofnucleic acid joined together by means of molecular biologicaltechniques. The term “recombinant” when made in reference to a proteinor a polypeptide refers to a protein molecule that is expressed using arecombinant nucleic acid molecule.

The term “isolated” when used in relation to a polynucleotide, refers toa nucleic acid sequence that is identified and separated from at leastone contaminant nucleic acid with which it is ordinarily associated inits natural source. Isolated nucleic acid molecule is present in a formor setting that is different from that in which it is found in nature.In contrast, non-isolated nucleic acids, such as DNA and RNA, are foundin the state they exist in nature. For example, a given DNA sequence(e.g., a gene) is found on the host cell chromosome in proximity toneighboring genes; RNA sequences, such as a specific mRNA sequenceencoding a specific protein, are found in the cell as a mixture withnumerous other mRNAs that encode a multitude of proteins. However,isolated nucleic acid molecule encoding a particular protein includes,by way of example, such nucleic acid in cells ordinarily expressing theprotein, where the nucleic acid is in a chromosomal location differentfrom that of natural cells, or is otherwise flanked by a differentnucleic acid sequence than that found in nature. The isolated nucleicacid may be present in single-stranded or double-stranded form. When anisolated nucleic acid is to be utilized to express a protein, thepolynucleotide will contain at a minimum the sense or coding strand(i.e., the polynucleotide may be single-stranded), but may contain boththe sense and anti-sense strands (i.e., the polynucleotide may bedouble-stranded).

The term “purified” refers to molecules, including nucleic or amino acidsequences that are removed from their natural environment isolated orseparated. An “isolated nucleic acid sequence” is therefore a purifiednucleic acid sequence. “Substantially purified” molecules are at least60% free, at least 75% free, or typically at least 90%, 95% or 99% freefrom other components with which they are naturally associated. As usedherein, the terms “purified” and “to purify” also refer to the removalof contaminants from a sample. The removal of contaminating molecules,including proteins, results in an increase in the percent of polypeptideof interest in the sample. In another example, recombinant polypeptidesare expressed in bacteria, yeast, or mammalian host cells and thepolypeptides are purified by the removal of host cell proteins; thepercent of recombinant polypeptides is thereby increased in the sample.

Nucleic acid sequences corresponding to genes or encoding polypeptidesrelating to the present invention can be readily purchased or purifiedfrom a suitable source by standard techniques, or can be synthesizedchemically. The nucleic acids can be genomic DNA, RNA, cDNA preparedfrom isolated mRNA, or DNA amplified from a naturally occurring nucleicacid sequence by standard techniques. Alternatively, the known sequencesmay be used to prepare probes to obtain other nucleic acid sequencesencoding a Wnt7a polypeptide from various sources using standardtechniques. Suitable sources for obtaining the nucleic acids are thosecells or tissues which are known to express the proteins of interest,such as skeletal muscle tissue and other tissues with measurable Wnt7atranscripts. An example of suitable cells would be myoblasts whichexpress Wnt7a.

Polynucleotides encoding fragments or variants of the naturallyoccurring Wnt7a proteins can be constructed by deletion, addition,and/or substitution of one or more nucleotides within the codingsequence using standard techniques, such as site-directed mutagenesistechniques.

Specific initiation signals may be required for efficient translation ofcloned polynucleotide. These signals include the ATG initiation codonand adjacent sequences. In cases where an entire wild-type gene or cDNA,including its own initiation codon and adjacent sequences, is insertedinto the appropriate expression vector, additional translational controlsignals may not be needed. In other cases, exogenous translationalcontrol signals, including, perhaps, the ATG initiation codon, must beprovided. Furthermore, the initiation codon must be in phase with thereading frame of the desired coding sequence to ensure translation ofthe entire insert. The exogenous translational control signals andinitiation codons can be natural or synthetic. The efficiency ofexpression may be enhanced by the inclusion of appropriate transcriptionenhancer elements and/or transcription terminators (Bittner et al.(1987) Methods in Enzymol. 153, 516).

In some instances, it may be desirable to link the coding sequence of aparticular gene to an amino- or carboxyl-terminal epitope tag tofacilitate detection or purification of expressed protein. Suitableepitope tags may include, but are not limited to, haemagluttanin (HA),myc, FLAG, 6×His, V5, glutathione-S-transferase (GST), etc.

An “expression vector”, also known as an expression construct, is usedto introduce a specific gene into a target cell. Once the expressionvector is inside the cell, the protein that is encoded by the gene isproduced by the cellular-transcription and translation machinery. Thevector is frequently engineered to contain regulatory sequences that actas enhancer and promoter regions and lead to efficient transcription ofthe gene carried on the expression vector. The goal of a well-designedexpression vector is the production of large amounts of stable messengerRNA.

Suitable expression vectors include, but are not limited to, plasmids,phagemids, viral particles and vectors, phages and the like. The entireexpression vector, or a part thereof, can be integrated into the hostcell genome. In some circumstances, it is desirable to employ aninducible expression vector as are known in the art, e.g. the LACSWITCHInducible Expression System (Stratagene, LaJolla, Calif.). Suitableexpression vectors may comprise promoters for driving expression in aparticular host cell. Some expression vectors may comprise a CMVpromoter. The expression vectors may be, for example, pCMV orpCMV-Sport6.

Those skilled in the field of molecular biology will understand that awide variety of expression systems can be used to provide therecombinant polypeptide or peptide. The polypeptide or peptide can beproduced in a prokaryotic host (e.g., E. cold or B. subtilis) or in aeukaryotic host (e.g., Saccharomyces or Pichia; mammalian cells, such asCOS, NIH 3T3, CHO, BHK, 293, or HeLa cells, insect cells, or plantcells). The methods of transformation or transfection and the choice ofexpression vector will depend on the host system selected and can bereadily determined by one skilled in the art. Transformation andtransfection methods are described, for example, in Ausubel et al.(1994) Current Protocols in Molecular Biology, John Wiley & Sons, NewYork; and various expression vectors may be chosen from those provided,e.g. in Cloning Vectors: A Laboratory Manual (Ponwels et al., 1985,Supp. 1987) and by various commercial suppliers.

In addition, a host cell may be chosen which modulates the expression ofthe inserted sequences, or modifies and processes the gene product in aspecific, desired fashion. Such modifications (e.g. glycosylation) andprocessing (e.g. cleavage) of protein products may be important for theactivity of the protein. Different host cells have characteristic andspecific mechanisms for the post-translational processing andmodification of proteins and gene products. Appropriate cell lines orhost systems can be chosen by one skilled in the art to ensure thecorrect modification and processing of the expressed heterologousprotein. The host cells harbouring the expression vehicle can becultured in conventional nutrient media adapted as needed for activationof a chosen gene, repression of a chosen gene, selection oftransformants, or amplification of a chosen gene according to knownprocedures.

In the context of the present invention, “oligonucleotide modulators”are oligonucleotide-based inhibitors or activators that are targeted toone or more components of the Wnt7a-PCP signaling pathway genes, orgenes encoding activators or effectors of the PCP pathway.Oligonucleotide modulators may, for example, include antisenseoligonucleotides, short interfering RNA (siRNA) molecules, ribozymes andtriple helix-forming oligonucleotides. RNA interference mediated bysiRNAs is known in the art to play an important role inpost-transcriptional gene silencing [Zamore, Nature Struc. Biol.,8:746-750 (2001)]. In nature, siRNA molecules are typically 21-22 basepairs in length and are generated when long double-stranded RNAmolecules are cleaved by the action of an endogenous ribonuclease.Recently, it has been demonstrated that transfection of mammalian cellswith synthetic siRNA molecules having a sequence identical to a portionof a target gene leads to a reduction in the mRNA levels of the targetgene. For example, Vangl2 expression is inhibited with siRNA, whichthereby inhibits symmetric cell division and results in an increase inasymmetric divisions. Oligonucleotide modulators can be prepared byconventional techniques well-known to those skilled in the art. Forexample, the oligonucleotides can be prepared using solid-phasesynthesis using commercially available equipment, such as the equipmentavailable from Applied Biosystems Canada Inc. (Mississauga, Canada).Alternatively, the oligonucleotide modulators can be prepared byenzymatic digestion and/or amplification of the naturally occurringtarget gene or mRNA, or of cDNA synthesized from the mRNA, usingstandard techniques known in the art. When the oligonucleotideinhibitors comprise RNA, they can be prepared by in vitro transcriptionmethods also known in the art. As indicated above, siRNA molecules canalso be conveniently prepared using commercially available in vitrotranscription kits. Oligonucleotides can also be prepared usingrecombinant DNA techniques.

As used herein, “primer” refers to an oligonucleotide containing two ormore deoxyribonucleotides or ribonucleotides, typically more than three,from which synthesis of a primer extension product can be initiated.Experimental conditions conducive to synthesis include the presence ofnucleoside triphosphates and an agent for polymerization and extension,such as DNA polymerase, and a suitable buffer, temperature and pH.

With reference to the polypeptide and polynucleotide sequences definedherein, the term “substantially identical” in reference to sequenceidentity means at least 70%, preferably at least 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity. By“identity” is meant the number of conserved amino acids or nucleotidesas determined by standard alignment algorithms or programs known in theart, used with default parameters established by each supplier. It willbe understood that the degree of identity may be represented by a rangedefined by any two of the values listed above or any value therebetween.Identity may be assessed, for example, over at least about 50, 100, 200,300, or more contiguous amino acids, or at least about 50, 100, 200,300, 500, 750, 1000 or more nucleotides, or may be assessed over thefull length of the sequence. The terms “homology” and “identity” areoften used interchangeably. Methods for determining % identity or %homology are known in the art and any suitable method may be employedfor this purpose. In general, sequences are aligned so that an optimizedmatch is obtained. Examples of an algorithm that is suitable fordetermining percent sequence identity is algorithms such as the BLASTalgorithm, as is well known to those skilled in the art. Software forperforming BLAST analyses is publicly available through the NationalCenter for Biotechnology Information (http://www.ncbi.nlm.nih.gov).Other commercially or publicly available programs include the DNAStarMegAlign program (Madison, Wis.) and the University of WisconsinGenetics Computer Group (UWG) Gap program (Madison Wis.).

As used herein, the term at “least 90% identical” would refer to percentidentities from 90 to 99.99% relative to a reference polynucleotide orpolypeptide. Identity at a level of 90% or more is indicative of thefact that, assuming for exemplification purposes a test and referencepolynucleotide or polypeptide length of 100 nucleotides or amino acidsare compared, no more than 10% of the respective nucleotides or aminoacids in the test polypeptide would differ from corresponding alignedpositions of the reference nucleotides/polypeptides. Differences may berepresented as point mutations randomly distributed over the entirelength of an polynucleotide or amino acid sequence or may be clusteredin one or more locations of varying length up to the maximum allowable,e.g. 10 of 100 nucleotide/amino acid differences for the above “at least90% identity” example. Differences may be defined as nucleic acid oramino acid substitutions or deletions.

Substantially identical nucleic acid molecules would hybridize typicallyat moderate stringency or at high stringency conditions along the lengthof the nucleic acid or along at least about 70%, 80%, 85%, 90%, 95%,96%, 97%, 98%, 99% of the full length nucleic acid molecule of interest.In the case of coding sequences, also contemplated are nucleic acidmolecules that contain degenerate codons in place of codons in thehybridizing nucleic acid molecule.

As used herein, “domain” refers to a portion of a molecule, e.g.,polypeptide or the encoding polynucleotide, that is structurally and/orfunctionally distinct from other portions of the molecule. For example,Fzd7 comprises a putative cysteine-rich Wnt-binding domain comprisingresidues 45 to 169 of the polypeptide shown as SEQ ID NO: 6 (mouse) andSEQ ID NO: 8 (human). Based on homology exhibited between human/mouseFzd7 and mouse Fzd8 and Fzd3 (for which crystal structures of therespective WNT-binding domain have been reported), residues 56, 58-60,and 62-64 of human/mouse Fzd7 may be particularly important forinteraction with Wnt7a. As another example, the final four residues ofeach of SEQ ID NOs: 6 and 8 encode a putative PDZ-binding motif(specifically residues 569-572 of SEQ ID NO: 6 and residues 571-574 ofSEQ ID NO: 8) (see, for example, Dann et al. Nature, 412, Jul. 5, 2001,p. 86-90).

As used herein, “gene therapy” includes both ex vivo and in vivotechniques. Thus host cells can be genetically engineered ex vivo with apolynucleotide, with the engineered cells then being provided to apatient to be treated. Delivery of the active agent in vivo may involvea process that effectively introduces a molecule of interest (e.g.Wnt-7A polypeptide or other activator of PCP-signaling) into the cellsor tissue being treated. In the case of polypeptide-based active agents,this can be effected directly or, alternatively, by transfectingtranscriptionally active DNA into living cells such that the activepolypeptide coding sequence is expressed and the polypeptide is producedby cellular machinery. Transcriptionally active DNA may be deliveredinto the cells or tissue, e.g. muscle, being treated using transfectionmethods including, but not limited to, electroporation, microinjection,calcium phosphate coprecipitation, DEAE dextran facilitatedtransfection, cationic liposomes and retroviruses. In certainembodiments, the DNA to be transfected is cloned into a vector. Suchvectors may include plasmids effective for delivery and expression ofthe DNA within a host cell. Such vectors may include but are not limitedto plasmids derived from human cytomegalovirus (hCMV) or other suitablepromotors such as hPGK-1 or hACT.

Alternatively, cells can be engineered in vivo by administration of thepolynucleotide using techniques known in the art. For example, by directinjection of a “naked” polynucleotide (Feigner and Rhodes, (1991) Nature349:351-352; U.S. Pat. No. 5,679,647) or a polynucleotide formulated ina composition with one or more other agents which facilitate uptake ofthe polynucleotide by the cell, such as saponins (see, for example, U.S.Pat. No. 5,739,118) or cationic polyamides (see, for example, U.S. Pat.No. 5,837,533); by microparticle bombardment (for example, through useof a “gene gun”, Biolistic, Dupont); by coating the polynucleotide withlipids, cell-surface receptors or transfecting agents; by encapsulationof the polynucleotide in liposomes, microparticles, or microcapsules; byadministration of the polynucleotide linked to a peptide which is knownto enter the nucleus; or by administration of the polynucleotide linkedto a ligand subject to receptor-mediated endocytosis (see, for example,Wu and Wu, (1987) J: Biol. Chem. 262:4429-4432), which can be used totarget cell types specifically expressing the receptors.

Alternatively, a polynucleotide-ligand complex can be formed in whichthe ligand comprises a fusogenic viral peptide to disrupt endosomes,allowing the polynucleotide to avoid lysosomal degradation; or thepolynucleotide can be targeted for cell specific uptake and expressionin vivo by targeting a specific receptor (see, for example,International Patent Applications WO 92/06180, WO 92/22635, WO92/203167WO93/14188 and WO 93/20221). The present invention also contemplates theintracellular introduction of the polynucleotide and subsequentincorporation within host cell DNA for expression by homologousrecombination (see, for example, Koller and Smithies (1989) Proc. Natl.Acad. Sci. USA 86:8932-8935; Zijlstra et al. (1989) Nature 342:435-438).

The polynucleotide can be incorporated into a suitable expressionvector. A number of vectors suitable for gene therapy applications areknown in the art (see, for example, Viral Vectors: Basic Science andGene Therapy, Eaton Publishing Co. (2000)) and may be used. Theexpression vector may be a plasmid vector. Methods of generating andpurifying plasmid DNA are rapid and straightforward. In addition,plasmid DNA typically does not integrate into the genome of the hostcell, but is maintained in an episomal location as a discrete entityeliminating genotoxicity issues that chromosomal integration may raise.A variety of plasmids are now readily available commercially and includethose derived from Escherichia cold and Bacillus subtilis, with manybeing designed particularly for use in mammalian systems. Examples ofplasmids that may be used in the present invention include, but are notlimited to, the expression vectors pRc/CMV (Invitrogen), pCR2. 1(Invitrogen), pAd/CMV and pAd/TR5/GFPq (Massie et al., (1998)Cytotechnology 28:53-64). In an exemplary embodiment, the plasmid ispRc/CMV, pRc/CMV2 (Invitrogen), pAdCMV5 (IRB-NRC), pcDNA3 (Invitrogen),pAdMLP5 (IRB-NRC), or pVAX (Invitrogen).

Alternatively, the expression vector can be a viral-based vector.Examples of viral-based vectors include, but are not limited to, thosederived from replication deficient retrovirus, lentivirus, adenovirusand adeno-associated virus. Retrovirus vectors and adeno-associatedvirus vectors are currently the recombinant gene delivery system ofchoice for the transfer of exogenous genes in vivo, particularly intohumans. These vectors provide efficient delivery of genes into cells,and the transferred polynucleotides are stably integrated into thechromosomal DNA of the host. A major prerequisite for the use ofretroviruses is to ensure the safety of their use, particularly withregard to the possibility of the spread of wild-type virus in the cellpopulation. Retroviruses, from which retroviral vectors may be derivedinclude, but are not limited to, Moloney Murine Leulcemia Virus, spleennecrosis virus, retroviruses such as Rous Sarcoma Virus, Harvey SarcomaVirus, avian leulcosis virus, gibbon ape leukemia virus, humanimmunodeficiency virus, adenovirus, Myeloproliferative Sarcoma Virus,and mammary tumour virus. Specific retroviruses include pLJ, pZIP, pWEand pEM, which are well known to those skilled in the art.

The polynucleotide is usually incorporated into the vector under thecontrol of a suitable promoter that allows for expression of the encodedpolypeptide in viva. Suitable promoters which may be employed include,but are not limited to, adenoviral promoters, such as the adenoviralmajor late promoter, the E1A promoter, the major late promoter (MLP) andassociated leader sequences or the E3 promoter; the cytomegalovirus(CMV) promoter; the respiratory syncytial virus (RSV) promoter;inducible promoters, such as the MMT promoter, the metallothioneinpromoter; heat shock promoters; the albumin promoter; the ApoAIpromoter; human globin promoters; viral thymidine kinase promoters, suchas the Herpes Simplex thymidine kinase promoter; retroviral LTR, thehistone, pot III, and (pectin promoters; B 19 parvovirus promoter; theSV40 promoter; and human growth hormone promoters. The promoter also maybe the native promoter for the gene of interest. The selection of asuitable promoter will be dependent on the vector, the host cell and theencoded protein and is considered to be within the ordinary skills of aworker in the art.

The development of specialized cell lines (termed “packaging cells”)which produce only replication-defective retroviruses has increased theutility of retroviruses for gene therapy, and defective retroviruses arewell characterized for use in gene transfer for gene therapy purposes(for a review see Miller, A. D.; 1990) Blood 76:271). Thus, recombinantretrovirus can be constructed in which part of the retroviral codingsequence (gag, pot, env) has been replaced by subject polynucleotide andrenders the retrovirus replication defective. The replication defectiveretrovirus is then packaged into virions that can be used to infect atarget cell through the use of a helper virus by standard techniques.Protocols for producing recombinant retroviruses and for infecting cellsin vitro or in vivo with such viruses can be found in Current Protocolsin Molecular Biology, Ausubel, F. M. et al. (eds.), J. Wiley & Sons,(1989), Sections 9.10-9.14 and other standard laboratory manuals.Examples of suitable packaging virus lines for preparing both ecotropicand amphotropic retroviral systems include Crip, Cre, 2 and Am. Otherexamples of packaging cells include, but are not limited to, the PE501,PA317, I-2, yr-3S AM, PA12, T1 9-14X, VT-1 9-1 7-H2, ACRE, SCRIP,GP+E-86, GP+envAml2, and DAN cell lines as described in Miller, HumanGene Therapy, Vol. 1, pas. 5-14 (1990).

Furthermore, it has been shown that it is possible to limit theinfection spectrum of retroviruses and consequently of retroviral-basedvectors by modifying the viral packaging proteins on the surface of theviral particle (see, for example PCT publications WO93/25234 andWO94/06920). For instance, strategies for the modification of theinfection spectrum of retroviral vectors include: coupling antibodiesspecific for cell surface antigens to the viral env protein (Roux et al.(1989) PNAS 86:9079-9083; Julan et al. (1992) J. Gen Virol 73:3251-3255;and Goud et al. (1983) Virology 163:251-254); or coupling cell surfacereceptor ligands to the viral env proteins (Neda et al. (1991) J. BiolChem 266: 14143-14146). Coupling can be in the form of the chemicalcross-linking with a protein or other variety (for example, lactose toconvert the env protein to an asialoglycoprotein), as well as bygenerating fusion proteins (for example, single-chain antibody/envfusion proteins). This technique, while useful to limit or otherwisedirect the infection to certain tissue types, can also be used toconvert an ecotropic vector in to an amphotropic vector.

Moreover, use of retroviral gene delivery can be further enhanced by theuse of tissue- or cell-specific transcriptional regulatory sequenceswhich control expression of the polynucleotides contained in the vector.

Another viral vector useful in gene therapy techniques is anadenovirus-derived vector. The genome of an adenovirus can bemanipulated such that it encodes and expresses a gene product ofinterest but is inactivated in terms of its ability to replicate in anormal lytic viral life cycle. See for example Beriner et al. (1988)BioTechniques 6:616; Rosenfeld et al. (1991) Science 252:431-434; andRosenfeld et al. (1992) Cell 68:143-155. Suitable adenoviral vectorsderived from the adenovirus strain Ad type 5 dl 324 or other strains ofadenovirus (for example, Adz, Ad3, Adz etc.) are well known to thoseskilled in the art. Recombinant adenoviruses can be advantageous incertain circumstances in that they can be used to infect a wide varietyof cell types, including peripheral nerve cells. Furthermore, the virusparticle is relatively stable and amenable to purification andconcentration, and as above, can be modified so as to affect thespectrum of infectivity. Additionally, introduced adenoviral DNA (andforeign DNA contained therein) is not integrated into the genome of ahost cell but remains episomal, thereby avoiding potential problems thatcan occur as a result of insertional mutagenesis in situations whereintroduced DNA becomes integrated into the host genome (for example,retroviral DNA). Moreover, the carrying capacity of the adenoviralgenome for foreign DNA is large (up to 8 kilobases) relative to othergene delivery vectors (Berliner et al. cited supra; Haj-Ahmand andGraham (1986) J. Virol. 57:267). Most replication-defective adenoviralvectors currently in use and contemplated by the present invention aredeleted for all or parts of the viral E2 and E3 genes but retain as muchas 80% of the adenoviral genetic material (see, e.g., Jones et al.(1979) Cell 16:683; Berliner et al., supra; and Graham et al. in Methodsin Molecular Biology, E. J. Murray, Ed. (Humane, Clifton, N.J., 1991)vol. 7. pp. 109-127). Generation and propagation ofreplication-defective human adenovirus vectors requires a unique helpercell line. Helper cell lines may be derived from human cells such ashuman embryonic kidney cells, muscle cells, hematopoetic cells or otherhuman embryonic mesenchymal or epithelial cells. Alternatively, thehelper cells may be derived from the cells of other mammalian speciesthat are permissive for human adenovirus, i.e. that provide, in bans, asequence necessary to allow for replication of a replication-deficientvirus. Such cells include, for example, 293 cells, Vero cells or othermonkey embryonic mesenchymal or epithelial cells. The use of non-humanadenovirus vectors, such as porcine or bovine adenovirus vectors is alsocontemplated. Selection of an appropriate viral vector and helper cellline is within the ordinary skills of a worker in the art.

As used herein, “subject” may be a mammalian subject, for example, butnot limited to mouse, cow, sheep, goat, pig, dog, cat, rat, rabbit,primate, or human.

A “pharmaceutical composition”, often used interchangeably withcomposition, includes at least one active agent for carrying out adesired effect. The pharmaceutical composition further comprises one ormore physiologically acceptable diluents, carriers or excipients.Pharmaceutical compositions and methods of preparing pharmaceuticalcompositions are known in the art and are described, for example, in“Remington: The Science and Practice of Pharmacy” (formerly “RemingtonsPharmaceutical Sciences”); Gennaro, A., Lippincott, Williams & Wilkins,Philadelphia, Pa. (2000).

A “cell composition” is a composition that contains cells together withone or more physiologically acceptable diluents, carriers or excipients.The cell composition may further comprise one or more active agents. Insome cases, the cells may be transformed to express a gene or protein ofinterest.

A “stem cell composition” is a composition that contains stem cellstogether with one or more physiologically acceptable diluents, carriersor excipients. The stem cell composition may comprise one or more activeagents, such as a stem cell modulator. In some cases, the stem cells maybe transformed to express a gene or protein of interest.

An “effective amount” is an amount sufficient to achieve a beneficial ordesired result. An effective amount may be effective amount in vitro orin vivo. In vivo, an effective amount may also be referred to as a“therapeutically effective amount”, which can be administered to apatient in one or more doses. In terms of treatment of disease ordamage, an effective amount may be an amount that is sufficient topalliate, ameliorate, stabilize, reverse or slow the progression of thedisease or damage. The effective amount is generally determined by thephysician on a case-by-case basis and is within the skill of one in theart. Several factors are typically taken into account when determiningan appropriate dosage to achieve an effective amount. These factorsinclude age, sex and weight of the patient, the condition being treated,the severity of the condition and the form and effective concentrationof the antigen-binding fragment administered.

As used herein, the term “about” refers to a +/−5% variation from thenominal value. It is to be understood that such a variation is alwaysincluded in any given value provided herein, whether or not it isspecifically referred to.

Embodiments of the invention are included within the definitions above,which may be relied upon to define the invention.

In one aspect, there is provided a composition for modulating thedivision symmetry of a stem cell comprising as an active agent amodulator of planar cell polarity (PCP) signaling in the stem cell.Preferably, the stem cell is an adult stem cell, for example, asatellite stem cell.

In some embodiments, the active agent is an activator of PCP signalingcapable of promoting symmetrical division of the stem cell. An activatormay comprise one or a combination of molecules. Polypeptide and peptideactivators of the PCP signaling pathway include direct activators, aswell as activators that exert their activating effect by inhibiting theactivity or expression of proteins that inhibit Wnt7a signaling, i.e.indirect activators.

The compositions described herein are useful in vitro or in vivo topromote stem cell expansion. It was demonstrated that activation of thePCP pathway, or components thereof, in satellite stem cells promotessymmetrical stem cell division, largely expanding the stem cell poolwithout affecting the rate of cell division.

The active agent may, for example, comprise a small molecule, apolynucleotide, a peptide, a polypeptide, a macromolecule, or acombination thereof.

The components of the PCP pathway, including Wnt7a and Fzd7, tend to behighly conserved across species. Therefore, polypeptides andpolynucleotides derived from various species are contemplated within thescope of the invention so long as they have the desired characteristicsand activity.

In some embodiments, the active agent comprises a peptide or polypeptidecapable of binding to and/or activating Fzd7 on the stem cell. Fzd7comprises a putative cysteine-rich Wnt-binding domain comprisingresidues 45 to 169 of the polypeptide shown as SEQ ID NO: 6 (mouse) andSEQ ID NO: 8 (human). Based on homology exhibited between human/mouseFzd7 and mouse Fzd8 and Fzd3 (for which crystal structures o therespective Wnt-binding domain have been reported), residues 56, 58-60,and 62-64 of human/mouse Fzd7 may be particularly important forinteraction with Wnt7a. Thus, a polypeptide capable of activating Fzd7may comprise a polypeptide capable of binding to a Wnt-binding domain ofFzd7.

In some embodiments, the active agent is a Wnt7a polypeptide or anactive analogue, variant, fragment, or derivative thereof capable ofbinding to and activating Fzd7. Exemplary Wnt7a polypeptides are shownin FIG. 22 (SEQ ID NO: 2) and 24 (SEQ ID NO: 4), which are mouse anhuman sequences, respectively.

In some embodiments, the active agent is a polypeptide having a sequenceof a Wnt7a polypeptide, or a sequence that is at least about 70%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to aWnt7a polypeptide. In some embodiments the sequence of the Wnt7apolypeptide comprises SEQ ID NO: 2 or SEQ ID NO: 4.

In some embodiments, the active agent is a Wnt7a polypeptide having anamino acid sequence comprising SEQ ID NO: 2 or SEQ ID NO: 4, or asequence that is at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 2 or SEQ ID NO: 4.

In some embodiments, the % identity is assessed over at least about 50,100, 200, 300, or more contiguous amino acids. In some embodiments, the% identity is assessed over the full length of the mature peptidesequence.

In certain embodiments, the active agent comprises a Wnt7a polypeptide.In some embodiments, the Wnt7a polypeptide is a human Wnt7a polypeptide.In some embodiments, the Wnt7a polypeptide is a murine Wnt7apolypeptide. Other species are also contemplated.

In some embodiments, the Wnt7a polypeptide has an amino acid sequencecomprising or consisting of SEQ ID NO: 2 or SEQ ID NO: 4.

In some embodiments, the active agent comprises an isolatedpolynucleotide encoding a peptide or polypeptide capable of binding toand/or activating Fzd7. The peptide or polypeptide capable of binding toand/or activating Fzd7 may be as described above. Thus, in someembodiments, the polynucleotide encodes a Wnt7a polypeptide or an activeanalogue, variant, fragment, or derivative thereof capable of binding toand activating Fzd7.

Exemplary Wnt7a polynucleotides are shown in FIG. 21 (SEQ ID NO: 1) and23 (SEQ ID NO: 3), which are mouse an human sequences, respectively.

In some embodiments, the active agent comprises a polynucleotideencoding a polypeptide having an amino acid sequence comprising SEQ IDNO: 2 or SEQ ID NO: 4, or a sequence that is at least about 70%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQID NO: 2 or SEQ ID NO: 4.

In some embodiments, the active agent comprises a polynucleotide havingan amino acid sequence comprising SEQ ID NO: 1 or SEQ ID NO: 3, or asequence that is at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1 or SEQ ID NO: 3.

In some embodiments, the % identity is assessed over at least about 50,100, 200, 300, 500, 750 or 100 or more contiguous nucleotides. In someembodiments, the % identity is assessed over the full length of thepolynucleotide.

In some embodiments, the polynucleotide comprises a Wnt7a polynucleotidesequence comprising or consisting of SEQ ID NO: 1 or SEQ ID NO: 3.

In some embodiments, the active agent is a small molecule capable ofbinding to and/or activating Fzd7.

In some embodiments, the active agent is a polynucleotide or polypeptidecapable of increasing expression of Fzd7 on the stem cell. In someembodiments, the active agent comprises a Fzd7 polynucleotide. ExemplaryFzd7 polynucleotides are shown in FIGS. 25 (SEQ ID NO 5) and 27 (SEQ IDNO: 7). In some embodiments, the active agent comprises a polynucleotidehaving an amino acid sequence comprising SEQ ID NO: 6 or SEQ ID NO: 8,or a sequence that is at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 6 or SEQ ID NO:8.

In some embodiments, the polypeptides correspond to modulators of Wnt7asignaling, for example, Fzd7, Fzd3, Celsr1, Celsr2, Celsr3, Dvl1, Dvl2,Dvl3, Pk1, Pk2, Pk3, Pk4, Rac/RhoA, Vangl1, Vangl2, Syndecan 4 (Syn4)and α7-β1-integrin, or active fragments, variants or derivativesthereof.

In some embodiments, the active agent comprises a polynucleotide orpolypeptide capable of modulating a downstream effector molecule in thePCP pathway to thereby promote or inhibit symmetrical cell division.Exemplary polarity effectors that may be modulated to affect celldivision decisions in adult stem cells include Prickle, Flamingo(Celsr2), Disheveled (Dsh) or PTK7. Exemplary targets of modulation inthe PCP pathway include, but are not limited to, Fzd7, Vangl1, Vangl2,Dvl2, Dvl3, Pk1, Pk2, Celsr2 and α7-integrin.

In some embodiments, the active agent comprises a polynucleotide orpolypeptide capable of activating a downstream effector molecule in thePCP pathway to thereby promote symmetrical stem cell division. Thedownstream effector molecule may, for example be, Vangl2, α7-integrin,Prickle 1 or Celsr2.

In one embodiment, the effector molecule is Vangl2. The active agentmay, for example, be a polypeptide or polynucleotide capable of inducingexpression or polarized redistribution of Vangl2 in the cell membrane.

In some embodiments, the active agent may comprise a polynucleotideencoding a Vangl2 polypeptide. Exemplary Vangl2 polypeptides are shownin FIGS. 30 (SEQ ID NO: 10) and 32 (SEQ ID NO: 12). In some embodiments,the Vangl2 polypeptide has substantially identical activity to thewild-type protein.

In some embodiments, the composition additionally comprise one or morestem cell modulators. The stem cell modulator may, for example, promoteone more of stem cell proliferation, differentiation, lineagecommitment, or terminal differentiation of committed progenitor cells.

In some embodiments, the modulator increases the rate of stem celldivision. Any suitable activator of stem cell division rate can be used,such as a suitable growth factor. Known growth factors include FGF, HGFand SDF. In some embodiments, a growth factor that increases stem celldivision rate without promoting differentiation is selected.

In some embodiments, the modulator is one that increases proliferationin a population of expanding stem cells, or one that promotesdifferentiation in a population of stem cells that have been previouslyexpanded by treatment with Wnt7a.

In some embodiments, a stem cell modulator promotes stem cell survival.Exemplary compounds that enhance the survival of the stem cells wouldinclude, for example, a sonic hedgehog (Shh) protein.

In some embodiments, a stem cell modulator is an inhibitor of canonicalWnt/β-catenin signaling.

In one embodiment, there is provided a composition for enhancing tissueformation, regeneration, maintenance or repair in a mammal comprising asan active agent (a) a Wnt7a polypeptide or an active variant, fragment,analogue or derivative thereof capable of binding to and activatingFzd7, or (b) a polynucleotide encoding a Wnt7a polypeptide or an activevariant, fragment, analogue or derivative thereof capable of binding toand activating Fzd7.

In another embodiment, there is provided a composition for promotingsymmetrical stem cell division comprising as active agent one or moreactivators of the Fzd7 receptor, wherein the one or more activators mayinclude, but are not limited to, one or more small molecules, nucleicacids, polypeptides, peptides, macromolecules, antibodies or acombination thereof, that activate Fzd7 receptor in adult stem cells.

In some embodiments, the adult stem cells are satellite stem cells.

The compositions described herein may be used to deliver apolynucleotide of interest into a cell or tissue. The composition may beadministered in vitro, for example, to expand a population of stemcells, or in vivo, for example, in a gene therapy method.

The polynucleotide of interest may be delivered directly into a cell ortissue (e.g. naked). More typically, the polynucleotide will be clonedinto an expression vector capable of expressing the encoded polypeptide.A cell or tissue may therefore be transformed to express a polypeptideof interest. Any suitable transformation method known in the art may beemployed.

Various expression systems are known in the art and are publicallyavailable through a number of sources (e.g. Invitrogen, Clontech). Anysuitable expression vector may be used. In some embodiments, theexpression vector is a mammalian expression vector. In some embodiments,the expression vector is a plasmid. In some embodiments, the plasmid isan virally-derived plasmid. In some embodiments, the plasmid comprises aCMV promoter sequence. In some embodiments, the plasmid comprises pCMVor pCMV-Sport6. In one embodiment, the plasmid is Wnt7a-CMV.

Gene expression may optionally be under the control of an induciblepromoter. Several inducible promoters are known in the art.

In some embodiments, the composition comprises an expression vectorcarrying a polynucleotide encoding a polypeptide capable of activatingPCP signaling in a stem cell, to thereby promote symmetrical stem cellexpansion.

In some embodiments, a cell or tissue is transformed to express amodulator of PCP signaling in a stem cell. A number of exemplarymodulators have been described above.

In some embodiments, a cell or tissue is transformed to overexpressWnt7a to thereby induce symmetrical division of a stem cell. Wnt7a maybe secreted from the transformed cell and may act on the cell from whichis it secreted or may act on a nearby stem cell. In some embodiments,the transformed cell is a helper cell that may be co-cultured orco-administered with the stem cell. The helper cell may also be aresident cell in a tissue that is transformed to overexpress Wnt7a oranother protein of interest.

In some embodiments, the helper cell is a myoblast or muscle celltransformed to overexpress Wnt7a.

In some embodiments, muscle tissue is transformed to overexpress Wnt7a.In some embodiments, the muscle tissue is skeletal muscle.Overexpression of Wnt7a expands the satellite stem cell pool in vivo,increases satellite cell numbers, and promotes muscle regeneration andrepair.

In some embodiments, a stem cell is transformed to overexpress Fzd7,Vangl2, α7-integrin, or another effector of PCP signaling in the stemcell.

In some embodiments, the composition comprises cells and may thereforebe a cell composition. For example, the composition may comprise ahelper cell. In some embodiments, the helper cell is transformed toexpress and secrete Wnt7a. In some embodiments, the helper cell is amyoblast or muscle cell.

In some embodiments, the composition comprises stem cells and istherefore a stem cell composition.

In some embodiments, stem cells may be expanded in vitro using a methodaccording to the invention and may subsequently be added to acomposition of the invention to form a stem cell composition. Forinstance, stem cells can be cultured and expanded in vitro using methodsof the invention and then administered to a subject as a therapeuticstem cell composition according to methods known to skilled persons.

In some embodiments, the composition comprises: a stem cell; and anactivator of PCP signaling in the stem cell. In some embodiments, thestem cell is a satellite stem cell.

In some embodiments, the composition comprises a stem cell transformedto express a polynucleotide of interest. Any suitable transformationmethod known in the art may be employed.

In one embodiment, there is provided a composition for enhancing tissueregeneration or repair comprising: a stem cell; and one or moreactivators or effectors of PCP signaling. Various activators andeffectors of PCP signaling have been described above.

In some embodiments, the composition comprises a stem cell transformedto overexpress an activator or effector of the PCP pathway, for example,Wnt7a, Fzd7 or Vangl2.

The composition may comprise a physiologically acceptable diluent,carrier or excipient. Methods of making various pharmaceuticalcompositions for various routes of administration are known in the artand are further describe in a later section.

In some embodiments, the composition is formulated for injection. Forinstance, the composition may be formulated for one or more ofintravenous injection, intramuscular injection, intracardiac injection,subcutaneous injection, or intraperitoneal injection. In one embodiment,the composition if for systemic injection. In one embodiment, thecomposition if for intramuscular injection.

In some embodiments, there is provided a use of a composition asdescribed herein for the manufacture of a medicament for promoting stemcell expansion. In some embodiments, there is provided a composition asdescribed herein for use in the manufacture of a medicament forpromoting stem cell expansion.

In some embodiments, there is provided a use of a composition asdescribed herein for the manufacture of a medicament for promotingmuscle formation, maintenance, repair, or regeneration of muscle in asubject in need thereof. In some embodiments, there is provided acomposition as described herein for use in the manufacture of amedicament for promoting muscle formation, maintenance, repair, orregeneration of muscle in a subject in need thereof.

In some embodiments, the composition is for promoting muscleregeneration or repair.

The composition may be administered in an effective amount, such as atherapeutically effective amount.

The invention provides for methods of modulating stem cells, inparticular, methods of modulating division symmetry of adult stem cells,such as satellite stem cells.

In some embodiments, there is provided a method for modulating divisionsymmetry of stem cells in vivo or in vitro comprising contacting thestem cells with a composition as described herein.

As described in the embodiments above, the composition comprises as anactive agent a modulator of planar cell polarity (PCP) signaling in thestem cell.

Is some embodiments the active agent is an activator of PCP signaling inthe stem cell and the method thereby promotes stem cell expansion. Suchmethods are useful, for example, for increasing the relative proportionof symmetrical to asymmetrical cell divisions in a population of stemcells in vivo or in vitro. Such methods are therefore useful forexpanding a population of stem cells in vivo or in vitro.

In some embodiments, the methods disclosed herein are capable ofpromoting symmetrical stem cell division without altering the rate ofstem cell division.

In some embodiments, the methods may be useful for promoting survival ofa population of stem cells.

In some embodiments, the methods are administered in vitro.

In some embodiments, the methods are administered in vivo. In someembodiments, the in vivo method comprises administering the compositionto a subject in need thereof.

In some embodiments, there is provided a method for expanding apopulation of satellite stem cells in vivo or in vitro comprisingcontacting the stem cells with an effective amount of a compositioncomprising (a) a Wnt7a polypeptide or an active variant, fragment,analogue or derivative thereof capable of binding to and activatingFzd7, or (b) a polynucleotide encoding a Wnt7a polypeptide or an activevariant, fragment, analogue or derivative thereof capable of binding toand activating Fzd7.

In some embodiments, the active agent is a Wnt7a polypeptide or ananalogue, derivative, variant or active fragment thereof.

In some embodiments, there is provided a method of promoting satellitestem cell expansion comprising contacting the satellite stem cell withWnt7a or an active fragment, variant, analogue or derivative thereofcapable of activating Fzd7.

In another embodiment, there is provided a method of increasing thenumber of satellite cells in a tissue, and thereby providing enhancedregeneration potential of the tissue, comprising contacting the stemcells with a composition as described herein.

In some embodiments, the methods of the invention are used in vivo fortreatment of resident stem cells in a tissue, e.g. resident satellitestem cells in muscle tissue.

In some embodiments, there are provided methods of promoting stem cellexpansion using compounds that activate Wnt7a, result in an increase ofendogenous Wnt7a or an increase in endogenous Wnt7a activity. Wnt7aactivators may be polypeptides or genes encoding polypeptides that actupstream of Wnt7a in vivo to upregulate expression or activity of Wnt7a,or they may be small molecule activators. Wnt7a activators may act at agenetic level, for example to upregulate the expression of a geneencoding Wnt7a, or they may act at the protein level to increase theactivity of a Wnt7a polypeptide or to decrease the activity of aninhibitor of Wnt7a. Wnt7a activators can be, for example, polypeptidesand peptides (or analogues, derivatives, variants or peptidomimeticcompounds corresponding to polypeptides, as described above),polynucleotides, oligonucleotides, antibodies or antibody fragments, ororganic or inorganic small molecules.

In some embodiments, the method may additionally comprise contacting thestem cell with one or more stem cell modulators, for example, amodulator that increases the rate of stem cell division or increasesstem cell survival.

In some embodiments, the method comprises administering cells to asubject. The cells may, for example, be administered simultaneously orsequentially with a composition described herein.

In some embodiments, the method comprises administering stem cells to asubject. The stem cells may, for example, be administered simultaneouslyor sequentially with a composition described herein that promotes stemcell expansion. For example, the stem cells may be administered prior toadministration of the composition (i.e. the composition may beadministered after a desired period). In some embodiments, thecomposition itself may comprise the stem cells to be administered.

Stem cells may be maintained and expanded in vitro for subsequentexperimental or therapeutic uses. In some embodiments, stem cells, e.g.satellite stem cells, are expanded in vitro and are subsequentlyadministered to a subject in need thereof. For instance, stem cells canbe cultured and expanded in vitro using methods of the invention andthen administered to a patient as a therapeutic stem cell compositionaccording to methods known to skilled persons.

In some embodiments, stem cells may be obtained from an individual andmaintained in culture. The population of cultured stem cells may betreated with Wnt7a, or another activator of PCP signaling, to promotesymmetrical expansion in vitro.

In some embodiments, the method may comprise administering helper cellsto a subject. The helper cells may, for example, be administeredsimultaneously or sequentially with the composition. In someembodiments, the composition itself may comprise helper cells.

The present invention also contemplates administration ofpolynucleotides encoding Wnt7a, a variant or active fragment thereof, oranother activator of PCP signaling, and optionally a stem cellmodulator, which then express the encoded product in vivo, by variousgene therapy methods known in the art.

In some embodiments, the method comprises transforming a cell or tissue.Some exemplary methods have been described previously above. Variousmethods of transformation are known to those of skill in the art.

In some embodiments, a cell or tissue is transformed to express Wnt7a,or an active fragment or variant thereof, which is then secreted andacts at the surface of stem cells by binding to the Fzd7 receptor.

In some embodiments, helper cells are transformed to overexpress Wnt7a,or an active fragment or variant thereof.

In some embodiments, stem cells are transformed to express Fzd7 or aneffector of PCP signaling, such as Vangl2.

In some embodiments, satellite stem cells are transformed to overexpressWnt7a, or an active fragment or variant thereof, or another activator ofPCP signaling. In one embodiment, satellite stem cells are transformedto overexpress Vangl2. In another embodiment, satellite stem cells aretransformed to overexpress Fzd7.

In some embodiments, stem cells are co-cultured with a differentiatedcell transformed to overexpress and secrete Wnt7a or another stimulatorof PCP signaling in the stem cell, e.g. an activator of Fzd7.

In one embodiment, satellite stem cells are co-cultured with musclecells transformed with CMV-Wnt7a to overexpress and secrete Wnt7a.

Any suitable expression vector may be used, including but not limited tothose described previously. Where in vivo methods are performed, cell-or tissue-specific vectors or promoters may also be used. In oneembodiment, the vector is a muscle-specific AAV vector. An induciblepromoter may optionally be used.

Polypeptide activators or effectors of PCP-signaling may be directlyintroduced into cells, bypassing the DNA transfection step. Means todirectly deliver polypeptides into cells include, but are not limitedto, microinjection, electroporation, cationic lipids and theconstruction of viral fusion proteins. Typically, transfection of asuitable expression system carrying a polynucleotide will be used.

The methods of promoting stem cell expansion can be used to stimulatethe ex vivo or in vitro expansion of stem cells and thereby provide apopulation of cells suitable for transplantation or administration to asubject in need thereof.

The stem cells to be administered may be treated with a stem cellmodulator, for example, a modulator that promotes survival of a stemcell. Sequential methods that promote expansion followed byproliferation and/or differentiation of stem cells are alsocontemplated. For example, a stem cell population may be expanded invitro by contacting the cells, directly or indirectly, with Wnt7a oranother activator of PCP signaling. The expanded population of cells maythen be treated with one or more stem cell modulators in vitro or invivo, e.g. that promote proliferation and/or differentiation of the stemcells in situ or promote stem cell survival. Alternatively, both stepsmay be conducted in vitro prior to administration of the cells to asubject.

In vitro is sometimes used interchangeably with ex vivo herein. For invivo and ex vivo transplant methods, the stem cells can be autologous,allogeneic or xenogeneic. In embodiments where stem cells from a donorsubject are transplanted into a recipient subject in need thereof,preferably, the donor and recipient are matched for immunocompatibility.For example, but not wishing to be limiting, it is preferable that thedonor and the recipient are matched for compatibility to the majorhistocompatibility complex (MHC) (human leukocyte antigen (HLA))-class I(e.g., loci A, B, C) and -class II (e.g., loci DR, DQ, DRW) antigens.Immunocompatibility between donor and recipient may be determinedaccording to methods generally known in the art (see, e.g., Charron, D.J., Curr. Opin. Hematol., 3: 416-422 (1996); Goldman, J., Curr. Opin.Hematol., 5: 417-418 (1998); and Boisjoly, H. M. et al., Opthalmology,93: 1290-1297 (1986)).

In one embodiment of the present invention, the gene therapy vector isan adenovirus-derived vector.

In one embodiment, Wnt7a-CMV is administered to a patient under thecontrol of a muscle-specific promoter or vector.

In some embodiments, the subject is a human.

The methods described herein have a number of applications. For example,the methods can be used in vitro to promote expansion of stem cellswherein the cells are destined for further in vitro use, for example,for research or diagnostic purposes. The methods can be used formaintaining stem cell cultures in vitro and also have potentialapplication in the development of new in vitro models for drug testingor screening.

The compositions and methods described herein are also useful forvarious therapeutic applications. In particular, the compositions andmethods described herein are useful for promoting tissue formation,regeneration, repair or maintenance in a subject in need thereof. Insome embodiments, the tissue is muscle. In some embodiments, the muscleis skeletal muscle.

Relevant therapeutic applications may pertain to situations where thereis a need to regenerate lost or damaged muscle tissue, for example,after chemotherapy or radiation therapy, after muscle injury, or in thetreatment or management of diseases and conditions affecting muscle. Insome embodiments, the disease or condition affecting muscle may includea wasting disease (e.g. cachexia, which may be associated with anillness such as cancer or AIDS), muscular attenuation or atrophy (e.g.sarcopenia, which may be associated with aging), ICU-induced weakness,prolonged disuse (e.g. coma, paralysis), surgery-induced weakness (e.g.following hip or knee replacement), or a muscle degenerative disease(e.g. muscular dystrophies). This list is not exhaustive.

In some embodiments, compositions and methods described herein areemployed where there is a need to prevent loss of tissue, as in wastingdiseases or atrophy.

In some embodiments, compositions and methods described herein areemployed where there is a need or desire to increase the proportion ofresident stem cells, or committed precursor cells, in a muscle tissue,for example, to replace damaged or defective tissue, or to preventmuscle atrophy or loss of muscle mass, in particular, in relation todiseases and disorders such as muscular dystrophy, neuromuscular andneurodegenerative diseases, muscle wasting diseases and conditions,atrophy, cardiovascular disease, stroke, heart failure, myocardialinfarction, cancer, HIV infection, AIDS, and the like.

In some embodiments, the methods can be used with satellite stem cellsin the treatment, management or prevention of degenerative muscledisorders.

In some embodiments, the compositions and methods are useful forpromoting muscle cell formation, for example, for repairing orregenerating dysfunctional skeletal muscle, for instance, in subjectshaving muscle degenerative diseases.

The subject may therefore have, be suspected of having, or be at risk ofat having skeletal muscle damage, degeneration or atrophy. The skeletalmuscle damage may be disease related or non-disease related. The humansubject may exhibit or be at risk of exhibiting muscle degeneration ormuscle wasting. The muscle degeneration or muscle wasting may be causedin whole or in part by a disease, for example aids, cancer, a musculardegenerative disease, or a combination thereof.

Muscle degeneration may be due to a muscle degeneration disease such asmuscular dystrophy.

Examples of muscular dystrophies include, but are not limited toDuchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD),myotonic dystrophy (also known as Steinert's disease), limb-girdlemuscular dystrophies, facioscapulohumeral muscular dystrophy (FSH),congenital muscular dystrophies, oculopharyngeal muscular dystrophy(OPMD), distal muscular dystrophies and Emery-Dreifuss musculardystrophy. See, e.g., Hoffman et al., N. Engl. J. Med., 318.1363-1368(1988); Bonnemann, C. G. et al., Curr. Opin. Ped., 8: 569-582 (1996);Worton, R., Science, 270: 755-756 (1995); Funakoshi, M. et al.,Neuromuscul. Disord., 9(2): 108-114 (1999); Lim, L. E. and Campbell, K.P., Cure. Opin. Neurol., 11(5): 443-452 (1998); Voit, T., Brain Dev.,20(2): 65-74 (1998); Brown, R. H., Annu. Rev. Med., 48: 457-466 (1997);Fisher, J. and Upadhyaya, M., Neuromuscul. Disord., 7 (1): 55-62 (1997).

In some embodiments, the muscular dystrophy is Duchenne musculardystrophy (DMD).

In some forms of urinary continence, the culprit muscle can be treatedwith a composition or method of the invention, for example, byelectroporation of the muscle. Thus, in one embodiment, the method isuseful for treating urinary incontinence.

In one aspect, there is provided a method for promoting muscleformation, regeneration or repair in a subject in need thereofcomprising administering to the mammal a composition comprising as anactive agent (a) a Wnt7a polypeptide or an active variant, fragment,analogue or derivative thereof capable of binding to and activatingFzd7, or (b) a polynucleotide encoding a Wnt7a polypeptide or an activevariant, fragment, analogue or derivative thereof capable of binding toand activating Fzd7.

In another aspect, there is provided a method for preventing musclewasting, atrophy or degeneration in a subject in need thereof comprisingadministering to the mammal a therapeutically effective amount of acomposition comprising (a) a Wnt7a polypeptide or an active variant,fragment, analogue or derivative thereof capable of binding to andactivating Fzd7, or (b) a polynucleotide encoding a Wnt7a polypeptide oran active variant, fragment, analogue or derivative thereof capable ofbinding to and activating Fzd7.

In some aspects, the compositions and methods described herein areuseful for promoting formation, maintenance, repair or regeneration ofskeletal muscle in a human subject in need thereof. In one aspect, thereis provided a method for enhancing tissue formation, regeneration,maintenance or repair in a mammal comprising administering to a subjectin need thereof a composition comprising as an active agent (a) a Wnt7apolypeptide or an active variant, fragment, analogue or derivativethereof capable of binding to and activating Fzd7, or (b) apolynucleotide encoding a Wnt7a polypeptide or an active variant,fragment, or derivative thereof capable of binding to and activatingFzd7.

The promotion of muscle cell formation can further be, in an embodiment,for preventing or treating muscle destruction or atrophy of a subject,e.g. in subjects with disuse atrophy or sarcopenia. In some embodiments,the compositions are used to treat or prevent atrophy and to maintainmuscle mass.

The promotion of muscle cell formation can also be, in an embodiment,for repairing damaged muscle tissue. In an alternative embodiment, thepromotion of muscle cell formation can be for increasing muscle mass ina subject.

In a further embodiment, damaged or dysfunctional muscle tissue may becaused by an ischemic event. For instance, the damaged muscle tissue maybe cardiac muscle damaged by a cardiovascular event such as myocardialinfarct, or heart failure.

In a further embodiment, damaged or dysfunctional muscle tissue may becardiac muscle. For instance, the damaged muscle tissue may be cardiacmuscle damaged by a cardiovascular event such as myocardial infarct, orheart failure, where the target stem cell would be a cardiac stem sell.In accordance with another aspect of the present invention, there isprovided a method of promoting cardiac stem cell expansion in a mammalcomprising administering to said mammal an effective amount of acomposition as described herein.

The compositions and methods described herein may be used in combinationwith other known treatments or standards of care for given diseases,injury, or conditions. For example, in the context of musculardystrophy, a composition of the invention for promoting symmetrical stemcell expansion can be administered in conjunction with such compounds asCT-1, pregnisone or myostatin. The treatments may be administeredtogether, separately or sequentially.

The present invention also contemplates methods of inhibitingsymmetrical stem cell expansion, for example, using compounds thatinhibit components of PCP signaling pathway. In another aspect, there isprovided a method for promoting asymmetrical stem cell divisioncomprising contacting a stem cell or population of stem cells with aninhibitor of PCP signaling.

In one aspect, there is provided a composition wherein the active agentis an inhibitor of PCP signaling capable of inhibiting symmetricaldivision of the stem cell. The inhibitor may, for example, be a peptide,polypeptide, polynucleotide or small molecule capable of directly orindirectly inhibiting PCP signaling via inhibition of Wnt7a, Fzd7, or aan effector molecule in the PCP pathway, e.g., Vangl2, α7-integrin,Prickle 1 or Celsr2.

In some embodiments, the inhibitor is a polynucleotide capable ofinhibiting expression of Wnt7a, Fzd7, or an effector molecule in the PCPpathway, e.g. siRNA or miRNA. In one embodiment, the inhibitor is Vangl2siRNA or Fzd7 siRNA.

Inhibition of PCP signaling in stem cells could be used, for example, inthe treatment of cancer, such as in the case of muscle tumorsrhabdomyosarcoma, or to treat diseases such as Fibrodysplasia OssificansProgressiva.

In some embodiments, the method may comprise contacting stem cells withan inhibitor of canonical Wnt/β-catenin signaling in the stem cell. Suchinhibition may further promote symmetrical stem cell division. In someembodiments, the composition may comprise a polynucleotide orpolypeptide inhibitor of canonical Wnt/β-catenin signaling in the stemcell.

The present invention further provides pharmaceutical compositionscomprising Wnt7a, an analogue, derivative, variant or active fragmentthereof, another activator or effector of PCP signaling, and apharmaceutically acceptable diluent or excipient. The pharmaceuticalcompositions may optionally further comprise one or more stem cellmodulators, one or more stem cells, or a combination thereof.Administration of the pharmaceutical compositions may be via a number ofroutes depending upon whether local or systemic treatment is desired andupon the area to be treated. Typically, the compositions areadministered systemically or locally to the area to be treated.

Administration may be topical (including ophthalmic and to mucousmembranes including vaginal and rectal delivery), pulmonary (e.g. byinhalation or insufflation of powders or aerosols, including bynebulizer), intratracheal, intranasal, epidermal and transdermal, oralor parenteral. Parenteral administration includes intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injection,for example, but not limited to intracardial injection or infusion, orintracranial, e.g. intrathecal or intraventricular administration. Insome embodiments, compositions are administered by injection orinfusion.

The compositions of the present invention may be delivered incombination with a pharmaceutically acceptable vehicle. Preferably, sucha vehicle would enhance the stability and/or delivery properties.Examples include liposomes, microparticles or microcapsules. In variousembodiments of the invention, the use of such vehicles may be beneficialin achieving sustained release of the active component. When formulatedfor parenteral injection, the pharmaceutical compositions are preferablyused in the form of a sterile solution, containing other solutes, forexample, enough saline or glucose to make the solution isotonic.

For administration by inhalation or insufflation, the pharmaceuticalcompositions can be formulated into an aqueous or partially aqueoussolution, which can then be utilized in the form of an aerosol. Fortopical use, the modulators or pharmaceutical compositions comprisingthe modulators can be formulated as dusting powders, creams or lotionsin pharmaceutically acceptable vehicles, which are applied to effectedportions of the skin.

In some embodiments, where the composition comprises a palmitoylatedprotein, such as Wnt7a, a lipid carrier may be employed.

The dosage requirements for the pharmaceutical compositions vary withthe particular compositions employed, the route of administration andthe particular subject being treated. Dosage requirements can bedetermined by standard clinical techniques known to a worker skilled inthe art. Treatment will generally be initiated with small dosages lessthan the optimum dose of each compound. Thereafter the dosage isincreased until the optimum effect under the circumstances is reached.In general, the pharmaceutical compositions are administered at aconcentration that will generally afford effective results withoutcausing any harmful or deleterious side effects. Administration can beeither as a single unit dose or, if desired, the dosage can be dividedinto convenient subunits that are administered at suitable timesthroughout the day.

When ex vivo methods of treating the stem cells are employed, the stemcells can be administered to the subject by a variety of procedures.Typically, administration of the stem cells is localized. The stem cellscan be administered by injection as a cell suspension in apharmaceutically acceptable liquid medium. Alternatively, the stem cellscan be administered in a biocompatible medium which is, or becomes insite a semi-solid or solid matrix. For example, the matrix maybe aninjectable liquid which forms a semi-solid gel at the site of tissuedamage or degeneration, such as matrices comprising collagen and/or itsderivatives, polylactic acid or polyglycolic acid, or it may compriseone or more layers of a flexible, solid matrix that is implanted in itsfinal boron, such as impregnated fibrous matrices. Such matrices areletdown in the art (for example, Gelfoam available from Upjohn,Kalamazoo, Mich.) and function to hold the cells in place at the site ofinjury, which enhances the opportunity for the administered cells toexpand and thereby for a reservoir of stem cells, to develop.

The stem cells may or may not be cryopreserved at some point.

In some embodiments, the stem cells are administered with a compound forpromoting stem cell expansion to minimize risk of stem cell depletionfollowing transplantation. In some embodiments, the transplanted stemcells have been transformed to overexpress an activator of PCPsignaling, such as Fzd7 or Vangl2.

In some embodiments, the stem cells are co-administered with musclecells or other satellite cells transformed to overexpress and secreteWnt7a.

In some embodiments, the stem cells are injected intramuscularly.

In a preferred embodiment, satellite stem cells or a compositioncomprising satellite stem cells is injected into muscle tissue,preferably in an area proximal to diseased, injured or damaged tissue.However, injection into the circulation or at a distal site is alsocontemplated. Intracardiac administration is also contemplated.

The present invention additionally provides for kits comprising acomposition as described herein together with one or more ofinstructions for use in promoting stem cell expansion or promotingtissue (e.g. muscle) formation, repair, regeneration, or maintenance.

The present invention additionally provides for therapeutic kitscontaining one or more modulators of the PCP pathway in stem cells, inpharmaceutical compositions.

The present invention additionally provides for therapeutic ordiagnostic kits containing Wnt7a, an analogue, derivative, variant oractive fragment thereof, or another activator of PCP signaling, andoptionally one or more stem cell modulators in pharmaceuticalcompositions.

Individual components of the kit would be packaged in separatecontainers and, associated with such containers, can be a notice in theform prescribed by a governmental agency regulating the manufacture, useor sale of pharmaceuticals or biological products, which notice reflectsapproval by the agency of manufacture, use or sale for human or animaladministration.

When the components of the kit may be provided in one or more liquidsolutions, the liquid solution can be an aqueous solution, for example asterile aqueous solution. In this case the container means may itself bean inhalant, syringe, pipette, eye dropper, or other such likeapparatus, from which the composition may be administered to a patient.

The components of the kit may also be provided in dried or lyophilizedform and the kit can additionally contain a suitable solvent forreconstitution of the lyophilized components. Irrespective of the numberor type of containers, the kits of the invention also may comprise aninstrument for assisting with the administration of the composition to apatient. Such an instrument may be an inhalant, syringe, pipette,forceps, measured spoon, eye dropper or any such medically approveddelivery vehicle.

Further, in addition to using the stem cells in transplants, stem cells,or compositions comprising stem cells may be used as a research tooland/or as part of a diagnostic assay or kit. Without wishing to belimiting a kit may comprise muscle stem cells, a promoter of Wnt 7asignaling, cell culture or growth medium, cell cryopreservation medium,one or more pharmaceutically acceptable delivery media, one or morenucleotide sequences or genetic constructs, one or more devices forimplantation or delivery of cells to a subject in need thereof,instructions for using, delivering, implanting, culturing,cryopreserving or any combination thereof the cells as described herein.

The ability of Wnt7a, analogues, derivatives, variants and activefragments thereof, or PCP activators or effectors, alone or incombination with other stem cell modulators, to promote stem cellexpansion can be tested in vitro or in vivo using standard techniquesincluding, but not limited to, those described herein. Inhibition ofstem cell expansion by one more inhibitors can also be measured in vitroor in vivo.

Candidate activators and inhibitors of stem cell expansion can also betested and identified using in vitro methods know to those skilled inthe art. Methods of maintaining stem cells in culture are known in theart (see, for example, Madlambayan, G. J., et al., (2001) J. Hematother.Stem Cell Res. 10, 481-492; Hierlihy, A. M., et al., (2002) FEBS Lett.530, 239-243; Asakura, A., et al., (2002) J Cell Biol. 159, 123-134).The stem cells can be cultured alone as a monoculture or they can beco-cultured with educator cells. Additional steps may be included in thescreening methods before, during, or after the culture period, such assteps to identify or isolate cell populations or otherwise contribute tothe success of the assay. For example, growth factors or other compoundsmay be employed to isolate and expand the stem cell population. EGF andFGF have been used for this purpose with neural stem cells as describedby Gritti et al (J. Neurosci. (1999) 19:3287-3297), and 13c1-2 has beenused in the isolation of “muscle stem cell” populations (see U.S. Pat.No. 6,337,184). In one embodiment, the culture medium used in DMEM plus10% FCS.

Various screening methods known in the art can be employed to identifycandidate activators of Wnt7a or another component of the PCP pathway,such as Vangl2. For example, activators that up- or down-regulate atarget gene can be identified by monitoring cells treated with thecandidate activator for an increase or decrease in the expression of thetarget gene. Methods such as Northern blot analysis, quantitative RT-PCRor microarray analysis can be used for this purpose. Alternatively, anincrease or decrease in the corresponding protein level can bemonitored, for example, by Western blot analysis.

For polypeptide or peptide activators (or analogues, derivatives,variants or peptidomimetic compounds corresponding to the polypeptides)that bind a specific protein, for example, Fzd7, the binding ability canbe determined using one of a variety of binding assays known in the art(see, for example, Coligan et al., (eds.) Current Protocols in ProteinScience, J. Wiley & Sons, New York, N.Y.).

For antibody or antibody fragment activators, various immunoassays canbe used.

In order to identify potential enhancers of symmetrical stem celldivision, a population of stem cells can be cultured and exposed tovarious test compounds. Furthermore, stem cells can be transfected toexpress various test genes of interest. Alternatively, stem cells can beco-cultured with “educator” cells, the educator cells are exposed to thetest compound(s) and at least one indicator of expansion is subsequentlymonitored in the stem cells. Educator cells may be exposed to the testcompound(s) prior to, or during, co-culture. Adult stem cells derivedfrom a variety of tissues can be used. Examples include, but are notlimited to, stem cells from cardiac or skeletal muscle, pancreatictissue, neural tissue, liver tissue or bone marrow, haematopoleticcells, myoblasts, hepatocytes, thymocytes, cardiomyocytes, and the like.Embryonic stem cells may also be used.

Generally, a compound is tested over a range of concentrations,typically about a 1000-fold range, and a suitable exposure protocol canbe readily established by one skilled in the art. When a co-culture isused, stem cell exposure to a compound can occur before, during or afterthe initial exposure of the stem cells to the educator cells.

Alternatively, when the test compound is a polynucleotide or a compoundencoded by a polynucleotide, such as a polypeptide or peptide, the stemcells can be transfected with the nucleic acid, or an expression vectorcomprising the polynucleotide, using standard methods described hereinand elsewhere, such that the test compound is produced endogenously.Additionally, the stem cells can be exposed to a test compound byco-culture of the stem cells with another cell line, which has beentransfected with the polynucleotide, or an expression vector comprisingthe polynucleotide, and which expresses the test compound.

As indicated above, it is further contemplated that the stem cells maynot be directly exposed to the compound. For example, an educator cellpopulation or a third cell type can be first treated with the compoundand subsequently co-cultured with the stem cells. Alternatively, thestem cells can be indirectly exposed by the addition of medium that hasbeen conditioned by such a cell population, but which is not itselfincluded in the co-culture. In addition, it is contemplated that thestem cells may be exposed to a compound that has been incorporated intoa non-liquid medium of the culture, for example, a solid, gel orsemi-solid growth support such as agar, a polymer scaffold, matrix orother construct.

Indicators of stem cell expansion may be monitored qualitatively orquantitatively and include, for example, changes in gross morphology,total cell number, histology, histochemistry or immunohistochemistry, orthe presence, absence or relative levels of specific cellular markers.The presence, absence or relative levels of cellular markers can beanalyzed by, for example, histochemical techniques, immunologicaltechniques, electrophoresis, Western blot analysis, FACS analysis, flowcytometry and the like. Alternatively the presence of mRNA expressedfrom the gene encoding the cellular marker protein can be detected, forexample, using PCR techniques, Northern blot analysis, the use ofsuitable oligonucleotide probes and the like.

For those cells treated with both Wnt7a (or another PCP activator oreffector) and a stem cell modulator, one or more indicators ofdifferentiation may also be monitored in the stem cell population aftertreatment with the modulator. Typically differentiation is monitored bychanges in gross morphology, as described above, or by the presence oflineage specific cellular markers, which can be analyzed using a numberof standard techniques as indicated above. Suitable lineage-specificcellular markers that can be monitored are known in the art.

Furthermore screening methods can be employed to identify new modulatorscapable of promoting stem cell expansion or promoting muscleregeneration and repair.

In one aspect of the invention, there is provided a method of screeningfor a compound useful in the repair or regeneration of muscle. Forexample, the method could comprise (a) providing a population ofsatellite stem cells; (b) treating the stem cells with a test compound;and (c) determining the proportion of symmetrical to asymmetricaldivisions of the treated stem cells compared to control, wherein aincrease in the proportion of symmetrical divisions compared to controlindicates that the compound is useful in the repair or regeneration ofmuscle.

In another aspect of the invention, there is provided a method ofscreening for a compound useful in the repair or regeneration of muscle.For example, the method could comprise (a) providing a population ofsatellite stem cells; (b) treating the stem cells with a test compound;and (c) determining whether the compound activates stimulates PCPsignaling in the treated stem cells, wherein a increase in PCP signalingindicates that the compound is useful in the repair or regeneration ofmuscle.

In some embodiments, the stimulation of PCP signaling occurs viaactivation of Fzd7. In some embodiments, the increase is an increase ofat least about 10%, 25%, 50%, 75% or greater.

Alternatively, the ability of Wnt7a, analogues, derivatives, variantsand active fragments thereof, or another activator or effector of PCPsignaling, to promote stem cell expansion, optionally in combinationwith one or more stem cell modulators, may be tested in vivo on residentstem cell populations in an appropriate experimental animal. Similarlyinhibitors may be tested in vivo. Typically, the test compound(s) areadministered directly to the tissue being investigated, for example, byinjection. After a suitable period of time, cells are harvested from theanimal and the stem cell population is analyzed as described above.

If desired, an inhibitor can also be tested, for example an inhibitor ofcanonical Wnt/8-catenin signaling. The compound and the inhibitor may beprovided to the stem cells concomitantly, or the compound may beprovided before or after the inhibitor.

In one embodiment, the ability of the compound(s) to promote stem cellexpansion is tested in vivo in murine skeletal muscle tissue. Theincrease in satellite stem cells isolated from treated mice is monitoredand compared to that in control mice, which are either untreated ortreated with a placebo, such as buffer or saline solution. In accordancewith one embodiment of the invention, a compound is considered topromote stem cell expansion when the satellite stem cell populationincreases by at least about 10%, e.g. compared to control. Anothermeasure is an increase in the proportion of symmetrical to asymmetricaldivisions. The increase in stem cells is measured over a time period ofat least 24 hours and more typically over a period of at least 96 hours.

The ability of Wnt7a, analogues, derivatives, variants and activefragments thereof, or other PCP activators or effectors to repairdamaged or dysfunctional tissue can be tested in a suitable animalmodel. Exemplary animal models are described in the Methods section. Forexample, the ability of the compound(s) and treatments to repair damagedmuscle tissue can be tested by administering the compound(s) ortreatments to mice exposed to freeze-induced or cardiotoxin inducedmuscle damage, and monitoring repair of the damaged muscle (see Megeneyet al., (1996) Genes Dev., 10:1173-1183; Asakura et al., (2000) J: CellBiol., 159:123-134).

It has been demonstrated that Fzd7 may be used as a marker for satellitestem cells when used in combination with at least one other stem cellmarker. This feature can be used to identify or isolate stem cells forsubsequent analysis, treatment, and/or transplantation. It was foundthat Fzd7 was particularly expressed on quiescent satellite stem cells.Therefore, Fzd7 could also be used as a marker of proliferative state ina stem cell.

It has also been demonstrated that a satellite stem cell may beidentified or isolated by selecting for the marker Pax7+ in combinationwith YFP− or Myf−. In one embodiment, there is provided a method ofidentifying or isolating satellite stem cells based on thecharacteristics YFP−/Pax7+. The method can be used to identify andisolate cells for subsequent analysis, treatment, and/ortransplantation.

SUMMARY OF EXPERIMENTAL FINDINGS

This section is a summary of the research findings described below inthe Examples. The scope of the invention is not in any way limited tothe content of this summary or the Examples that follow.

The present inventors previously identified a small population ofsatellite stem cells and reported that satellite cells represent aheterogeneous population of stem cells and progenitor cells. Thesatellite stem cells were shown to be capable of self-renewal andlong-term reconstitution of the satellite cell niche followingtransplantation (Kuang et al., 2007).

Through diligent research efforts, the present inventors have nowdetermined that the non-canonical Wnt receptor Fzd7 is specificallyexpressed in quiescent satellite cells (FIG. 4). Wnt7a was examined as acandidate ligand for Fzd7. We found by Real-time PCR andimmunohistochemistry that Wnt7a was markedly upregulated in newly formedmyofibers during regenerative myogenesis (FIG. 5B, 5E), and that theFzd7 receptor is necessary for Wnt7a binding at the surface of myogeniccells (FIG. 5C). Satellite stem cells undergo apical-basal asymmetriccell divisions to give rise to committed myogenic cells that expressMyf5, and to maintain their population through self-renewal.Alternatively, satellite stem cells can undergo planar symmetric celldivisions to drive expansion of their population (Kuang et al., 2007).Importantly, the present inventors found that recombinant Wnt7a proteindramatically stimulated the symmetric expansion of satellite stem cellsand that this expansion required Fzd7 and Vangl2 (FIGS. 6 and 8), bothcomponents of the planar cell polarity (PCP) signaling pathway.Moreover, Wnt7a induced polarized localization of Vangl2 at oppositepoles in pairs of dividing cells (FIG. 7), in a manner consistent withWnt7a activating PCP signaling. Over expression of Wnt7a during muscleregeneration resulted in an impressive enhancement of the regenerationprocess, generating more fibers of bigger caliber, independent of aneffect on myoblast proliferation or differentiation (FIG. 9).Importantly, Wnt7a over-expression resulted in a large expansion of thesatellite stem cell population, and Wnt7a loss resulted in impairedmaintenance of the satellite cell compartment (FIG. 10). These resultsprovide important new insights into the molecular regulation ofsatellite cell self-renewal, and for the first time implicate the Wntnon-canonical PCP pathway in the regulation of adult stem cell function.

The Wnt-PCP pathway plays a role in patterning by instituting polarityof cells within a tissue, such as with the organized orientation ofepithelial cells in Drosophila (Zallen, 2007). In vertebrates, PCPsignaling, and particularly its effecter Vangl2 (also known asStrabismus), is required for the polarization of stereociliary bundlesin the cochlea (Montcouquiol et al., 2003), for convergent extension(CE) movements regulating gastrulation and neurulation (Torban et al.,2004), neural tube closure (Torban et al., 2008), and in regulatingmyocyte orientation in the developing myotome (Gros et al., 2009).During zebrafish neurulation, loss of Vangl2 abrogates polarization ofneural keel cells by preventing re-intercalation of daughter cells intothe neuroepithelium, resulting in ectopic neural progenitor accumulation(Ciruna et al., 2006). Based on the findings presented herein, it is nowproposed that the symmetric expansion of satellite stem cells resultsfrom a PCP-mediated orientation of the axis of stem cell division. SincePCP is a positional signaling relying on the redistribution of effectorproteins, polarization of PCP core molecules on opposite poles of thedaughter cells allows both cells to maintain contact with the basallamina and thus preserve their orientation relative to the niche (FIG.7). Notably, Wnt7a induced polarized distribution of Vangl2 andα7-integrin (FIG. 7E). The upregulated and polarized localization ofα7-integrin allows both daughter cells to remain attached to the basallamina. By contrast, α7-integrin expression is reduced and evenlydistributed in apical-basal oriented cell divisions. Daughter cells thatare “pushed” towards the sarcolemma, thus losing contact with the basallamina, activate Myf5 transcription and commit to a progenitor state(Kuang et al., 2007). Therefore, these data suggest that the PCP pathwayintersects with the mechanisms that control apical-basal cell divisionsand commitment and function through a mechanism that promotes adhesionto the basal lamina.

The experiments suggest that polarized distribution of Vangl2 protein atthe poles of a couplet of daughter cells allows both cells to remainattached to the basal lamina, and therefore maintain a stem cell state,resulting in expansion of the stem cell population. Subsequent celldivisions will generate larger numbers of committed daughter cellsthrough apical-basal asymmetric divisions that will undergo normalexpansion and differentiation (FIG. 9). We previously noted that theproportion of Pax7⁺/Myf5⁻ satellite stem cells increased from 10% toabout 30% at 3 weeks following injury (Kuang et al., 2007), and we havenow demonstrated that overexpression of Wnt7a further increased thelevel to 50% (FIG. 10C, 10D). By contrast, satellite cell numbersdecreased by 36% in Wnt7a-deficient muscle following injury andregeneration (FIG. 10G). These data suggest that Wnt7a regulates thehomeostatic maintenance of the satellite stem cell pool by modulatingthe increase in satellite stem cell expansion during regenerativemyogenesis, and that basal levels of PCP signaling are insufficient tomaintain the satellite cell pool at normal levels.

Canonical Wnt-signaling plays a well-documented role in regulatingmyogenic growth and differentiation. The experiments described hereinindicate that activation of Wnt/β-catenin signaling using Wnt3a did notinterfere with satellite stem cell choice between commitment andsymmetric expansion (FIGS. 6B, 6D). Nevertheless, over-expression ofWnt3a in vivo appeared to impair regeneration, likely by promotingpremature differentiation and the formation of myofibers of reduced size(FIG. 9). Indeed, Wnt3a stimulation of satellite cells on singlemyofibers drove their differentiation as evidenced by significantincrease in the number of Pax7⁻/MyoD⁺ cells (not shown). However, Wnt3aexpression was not detected in undamaged or regenerating skeletal muscleby Real-Time PCR. Potentially, other upregulated Wnts, such as Wnt10a(FIG. 5B), may function to activate the Wnt/β-catenin signaling pathwayin myogenic cells. Furthermore, a large up-regulation of theWnt-inhibitors sFRPs was observed during the early stages of theregenerative process. This may represent a physiological feedback systemthat inhibits canonical Wnt signaling, allowing the proliferation ofmyogenic progenitors. Thus, it is hypothesized that inhibition ofWnt/β-catenin signaling would act to promote muscle regeneration.

In Xenopus embryos, the Vangl2 homolog Strabismus inhibits Wnt/β-cateninactivated transcription by competing for Disheveled (Park and Moon,2002). Thus, without wishing to be bound by theory, PCP signaling mayalso act to keep satellite stem cells in an uncommitted state byantagonizing canonical Wnt/β-catenin signaling. In Drosophila eyedevelopment, Frizzled (Fzd)/PCP signaling induces cell-fatespecification of the R3/R4 photoreceptors through regulation of Notchactivation in R4 (del Alamo and Mlodzik, 2006). This raises thepossibility that cross-talk between Frizzled/PCP and Notch pathways, aswell as Wnt/β-catenin pathways, act to coordinate satellite stem cellchoice between self-renewal/commitment versus expansion. Molecularcharacterization of satellite stem cells is providing important insightsinto the molecular mechanisms regulating their function. The presentidentification of a role for the Wnt7a/Fzd7/Vangl2 signal transductioncascade reveals an unanticipated role for the PCP pathway in regulatingthe symmetric expansion of satellite stem cells. This finding representsa significant advance in our understanding of satellite cell biology andmuscle regeneration. Future experiments will investigate both theutility of modulating the PCP pathway to augment muscle regenerationtowards ameliorating the loss of muscle function in neuromusculardisease.

EXAMPLES Example 1 Frizzled7 is Highly Expressed in Quiescent SatelliteStem Cells

Satellite cells are a heterogeneous population composed of stem cellsand committed progenitors. All satellite cells express Pax7 and markerssuch as CXCR4, however, the present inventors identified a subset ofabout 10% of Pax7+ cells that have never expressed Myf5 during theirdevelopmental history (FIG. 4A). This subset of Pax7⁺/Myf5⁻ satellitewas identified as a stem cell population within the satellite cell niche(Kuang et al., 2007). Towards performing gene expression analysis ofquiescent satellite stem cells, the inventors first developed animproved methodology for satellite cell isolation by fluorescenceactivated cell sorting (FACS), as described in the Methods section.FACS-purified cells (CD34⁺, α7-Integrin⁺, CD31⁻, CD45⁻, CD11 b⁻, Scat)(FIG. 11A), were >95% satellite cells as determined by Pax7 andSyndecan4 expression (FIG. 11B), and exhibited robust growth anddifferentiation potential in vitro (FIG. 11C).

FACS-purified satellite cells were further separated on the basis ofMyf5-conditional YFP fluorescence (FIG. 4B). In vitro cultured YFP⁻satellite cells gave rise to proliferating cells expressing Pax7, butnot YFP or Myf5 protein, when maintained at low density (FIG. 12), thusvalidating that YFP⁻ cells do not and have not expressed Myf5. Real-TimePCR analysis of freshly sorted cells confirmed Pax7 expression (FIG.4C), as well as several other satellite cell markers such as cMet,Syndecan4, Caveolin1 and α7-Integrin (not shown) in isolated YFP⁺ andYFP⁻ satellite cells. In addition, YFP and Myf5 transcripts weredetected in YFP⁺ satellite cells while virtually no YFP and Myf5expression (not significantly different from RT⁻ controls) were detectedin YFP⁻ satellite cells (FIG. 4C). Suppressive subtractive hybridization(SSH) of cDNAs (Diatchenko et al., 1997) was employed to identify genesexpressed specifically in quiescent Pax7+/Myf5− satellite stem cells.Notably, one of the differentially expressed clones encoded a fragmentfrom within the Frizzled7 (Fzd7) mRNA. Fzd7 is a G-protein-coupledtransmembrane Wnt receptor that belongs to a protein family encoded bymultiple genes (Egger-Adam and Katanaev, 2008). Real-time PCR analysisconfirmed that Fzd7 transcripts were abundantly expressed in YFP⁻satellite cells and only marginally detected in YFP⁺ satellite cells(FIG. 4C).

To confirm the differential expression suggested by Real-Time PCR, Fzd7protein expression was examined in myofibers fixed immediately followingisolation from extensor digitorum longus (EDL) muscles.Immunohistological analysis revealed that 12±3% of Pax7⁺ satellite cellsexpressed readily detectable levels of Fzd7 (n=3 mice, >150cells/mouse). Analysis of myofibers isolated from Myf5-Cre/ROSA26-YFPEDL muscles demonstrated that Fzd7 was specifically upregulated insatellite stem cells (Pax7⁺/Myf5⁻) that do not contain detectable levelsof YFP (FIG. 4D). However, culture of fibers in suspension for 2 daysresulted in upregulation of Fzd7 in virtually all satellite cells (99%,n=3 mice, ≥100 cells per mouse). Furthermore, examination ofregenerating myofibers from EDL muscle following cardiotoxin (CTX)induced damage of the tibialis anterior (TA) muscle (Kuang et al.,2007), revealed Fzd7 expression on doublets of Pax7⁺/Myf5⁻ andPax7⁺/Myf5⁺ cells (FIG. 4E).

Taken together, these results demonstrate that, in resting muscle, theWnt receptor Fzd7 is specifically expressed in quiescent satellite stemcells. However, Fzd7 is also upregulated in proliferating satellitecells and myoblasts. Thus, the Fzd7 receptor may be considered a markerof quiescent satellite stem cells in resting muscle, and may beparticularly useful as a marker of quiescent satellite stem cells incombination with other stem cells markers. The Fzd7 receptor may also beused as a target for purification of quiescent satellite stem cells, forinstance, employing antibodies reactive to Fzd7.

Example 2 Wnt Expression During Muscle Regeneration

The present inventors hypothesized that Wnt7a was a candidate ligand forFzd7 receptor. Coexpression of Fzd7 and Wnt7a during embryonicmyogenesis had been reported (Cossu and Borello, 1999) and Wnt7a hadbeen implicated as a regulator of embryonic and adult myogenesis (Chenet al., 2005; Polesskaya et al., 2003; Tajbakhsh et al., 1998).Real-Time PCR-Array analysis of freeze-injured TA muscle was employed todocument Wnt expression during regenerative myogenesis. Freeze injury ofmuscle was chosen because of the significantly reduced inflammatoryresponse relative to other methods such as cardiotoxin (CTX) injection.Changes in gene expression were analyzed at 3 days post-injury, duringthe acute phase of regeneration, where most of the Pax7⁺ cells areproliferating, and at 6 days post-injury, when satellite cells havereturned to a quiescent sub-laminar position (FIG. 5A).

At 3 days post-injury significant increases (as compared tocontralateral leg, n=3 mice, p<0.05) in 31 transcripts were detected,including those for multiple Wnts (Wnt-1, -2, -5b, -8b, -10a, -16a),Frizzled receptors and sFRP inhibitors. Notably, at 6 days post-injury asignificant increase (n=3 mice, p<0.05) was detected in the transcriptlevels for Wnt7a and Wnt10a, (FIG. 5B). Wnt3a levels were below thelimit of detection in the analyses at both 3 and 6 days of regeneration.Therefore, Wnt7a mRNA was markedly upregulated at the time whensatellite stem cells replenish the resident satellite cell pool.

To confirm Wnt7a up-regulation during muscle regeneration in anothermuscle injury model, immunohistochemical analysis of Wnt7a proteinexpression was performed on cryosections of CTX-injured TA (fixed 4 dayspost-injury) and the contralateral resting TA. In undamaged muscle,Wnt7a was not expressed at detectable levels (FIG. 5E, left). Bycontrast, Wnt7a was strongly upregulated in regenerating fibers (ofsmaller size than the intact fibers and containing Myogenin⁺ nuclei),and was not expressed by CD144⁺ endothelial cells (FIG. 5E, right).

To determine whether Wnt7a is a ligand for Fzd7, cultured satellitecell-derived myoblasts were incubated with recombinant human Wnt7aprotein for 30 minutes, washed, fixed and immunostained with anti-Wnt7aantibody. Cells incubated with BSA did not show membrane staining forWnt7a protein. By contrast, cells incubated with Wnt7a protein exhibitedimmunostaining on the membrane (FIG. 5C). Importantly, transfection ofFzd7 siRNA abrogated binding of Wnt7a (FIG. 2C). Fzd7 silencing waseffective, specific and did not significantly alter the other Frizzledtranscripts expressed in myoblasts (FIG. 14A). Taken together, thesedata indicate that Fzd7 is the receptor for Wnt7a in myogenic cells.

Wnt7a has been described as either a canonical (Hirabayashi et al.,2004) or non canonical Wnt (Kengaku et al., 1998), depending oncell-type and receptor context. To evaluate the possible function ofWnt7a as a canonical Wnt, satellite cell-derived myoblasts werestimulated with Wnt7a and Wnt3a proteins for 24 hours. Wnt3a activatesthe canonical Wnt pathway in myogenic cells (Brack et al., 2008), and inthe present experiment, Wnt3a stimulation resulted in increasedexpression of β-catenin/TCF target genes, for example a 5-fold and50-fold increase of Tcf7 and Axin2 mRNAs respectively, (n=5, p≤1.001).By contrast, Wnt7a stimulation did not result in any significant changein Tcf7 and Axin2 levels which were similar to BSA-treated samples (FIG.5D). In addition, Wnt3a but not Wnt7a stimulation robustly induced thestabilization and nuclear localization of activated-β-catenin (FIG. 13),and Wnt3a but not Wnt7a robustly activated the β-catenin luciferasereporter TOP-Flash in transient transfection experiments (not shown).

Taken together, these results indicate that Wnt7a is markedlyupregulated by newly formed myofibers during regenerative myogenesis,binds to the Fzd7 receptor at the surface of myogenic cells, and doesnot utilize the canonical Wnt/8-catenin signaling pathway.

Example 3 Symmetry of Satellite Stem Cell Divisions is Regulated byWnt7a-Frizzled7

The expression of Fzd7 in quiescent satellite stem cells and the markedupregulation in Wnt7a during muscle regeneration suggested thatWnt7a-Fzd7 signaling is involved in regulating muscle stem cellfunction. In addition, Wnt7a had no effect on the growth ordifferentiation of cultured primary myoblasts in vitro (FIG. 16).Therefore, to investigate the role of Wnt7a-Fzd7 signaling in satellitecells, the ability of recombinant Wnt7a to alter the ratio betweenasymmetric and symmetric cell divisions of satellite stem cells wasexaminted in vitro. Myofibers were isolated from Myf5-Cre/ROSA26-YFP EDLmuscle and cultured under non-adherent conditions. In the culturesystem, quiescent satellite cells become activated immediately followingmyofiber isolation. Satellite cells leave their niche, migrate acrossthe basal lamina, and undergo their first cell division in a synchronousfashion. Thus, the outcome of the first division was visualized byfixing and staining the myofibers after 42 hours of culture.Importantly, live imaging analysis confirms that satellite cells do notmove on myofibers before dividing and that scored cell doublets are ofclonal origin (Kuang et al., 2007).

Satellite stem cells (YFP⁻) either underwent a symmetrical cell divisionto give rise to two YFP⁻ daughter cells, or an asymmetric cell divisionto give rise to one YFP⁻ stem cell and one YFP⁺ committed precursor(FIG. 6A). When stimulated with Wnt7a, a dramatic increase in theproportion of symmetric cell divisions from 30% to 67% was observed(n=3, n≥152 pairs, p=0.009). By contrast, Wnt3a treatment did not induceany significant change (FIG. 6B). Therefore, Wnt7a stimulated anincrease in symmetric satellite stem cell divisions.

The experimental analysis suggested that Wnt7a specifically binds theFzd7 receptor (FIG. 5C, 5D). Therefore, to determine whether theinduction of symmetric stem cell divisions by Wnt7a required thepresence of Fzd7, Fzd7 knock-down experiments were performed on isolatedmyofibers. Immunostaining of treated fibers demonstrated extensivesilencing of Fzd7 expression after 42 hours (FIG. 5C). Importantly,siRNA-induced knock-down of Fzd7 resulted in a complete abrogation ofthe ability of Wnt7a to induce symmetric satellite stem cell divisions(n=3, ≥123 pairs, p≤1.02). By contrast, scrambled siRNA treatment didnot significantly affect this activity of Wnt7a (FIG. 6D). Consistentwith these results, the proportion of satellite stem cells (Pax7⁺/YFP⁻)after the second division (50 hours) was significantly increased by 13%after Wnt7a treatment (n=3, ≥1203 cells, p≤1.03) this resulting in anincrease in the number of stem cells per fibers (FIGS. 6E, 14B).Similarly, Fzd7 silencing efficiently blocked the effect of Wnt7astimulation. The total number of Pax7⁺ cells per fiber remained constantbetween each condition, confirming that Wnt7a does not effect cellproliferation or differentiation (FIG. 14C).

These results demonstrate that Wnt7a signals via Fzd7 to stimulatesymmetric satellite stem cell division and thus drive the expansion ofthe satellite stem cell pool.

Example 4 Role for the PCP Component Vangl2 in Satellite Stem CellSelf-Renewal

The analysis indicated that Wnt7a does not activate the canonicalWnt/β-catenin signaling pathway (FIG. 5D), and that Wnt7a signalsthrough the Fzd7 receptor to drive satellite stem cell symmetricdivisions (FIGS. 5C, 6B, 6D). Therefore, it was hypothesized that Wnt7aacts through Fzd7 to activate the PCP pathway and drive satellite stemcell expansion. To investigate if Wnt7a activates the PCP pathway, therelative transcript levels of a set of core PCP components (Seifert andMlodzik, 2007) were analyzed in myogenic cells. Interestingly, myoblastsexpressed significant levels of Dvl-2 and -3, Fzd-3 and -7, Pk-1 and -2and yang/4 and -2, and low levels of Celsr2. Other PCP component genestested were called absent with cut-off values over 30 cycles (FIG. 4A).In addition, cultured satellite stem cells (YFP⁻) expressedsignificantly higher levels of all PCP components (n=3, p<0.05), with amarked upregulation of Vangl2, consistent with a role for PCP signalingin regulating satellite stem cell function.

Vangl2 is a crucial regulator of PCP and non-canonical Wnt-signaling inDrosophila and vertebrates (Torban et al., 2004). In cells with activePCP signaling, Vangl2 protein is distributed at the poles at either endof the axis of polarization and this distribution is lost in PCP mutants(Montcouquiol et al., 2006). Vangl2 protein was not detected inquiescent satellite cells on isolated myofibers, but was upregulated inactivated satellite cells as they entered the cell cycle by 24 hours inculture. After 48 h, all Pax7⁺ activated satellite cells were alsopositive for Vangl2 (100%, n=3 mice, ≥100 cells per mouse) (FIG. 7B).The expression in satellite cells of Prickle1 and Celsr2 proteins thatinteract with Vangl2 in vivo was also confirmed (not shown).

Importantly, in the presence of Wnt7a, a significant proportion ofdividing doublets of satellite cells on cultured myofibers (29±4%, n=3,≥240 pairs, p<1.006) exhibited polarized localization of Vangl2 onopposite poles of the daughter cells (FIG. 7C, 7D). By contrast,following BSA (control) or Wnt3a-treatment, Vangl2 protein was uniformlydispersed in satellite cell doublets (90±2% and 89±2% respectively)(FIG. 7C, 7D). Moreover, double staining with anti-Vangl2 andanti-□α7-integrin antibodies revealed that Wnt7a appeared to induceenhanced membrane localization of Vangl2 and polarized distribution ofα7-integrin. This redistribution did not occur in untreated cells or incells undergoing apical-basal cell divisions (FIG. 7E). Taken together,these observations strongly support the assertion that Wnt7a induces aredistribution of the polarity effector Vangl2 and α7-integrin and theupregulated expression of α7-integrin at the poles of daughter cellsallows them to remain adherent with the basal lamina and to remain inthe stem cell niche.

To investigate the role of Vangl2 in satellite stem cell function, siRNAsilencing of Vangl2 was performed on single Myf5-Cre/ROSA26-YFPmyofibers stimulated with Wnt7a. Myofibers were first stained with Pax7and Syndecan4 antibodies to allow visualization of the plane ofsatellite cell division relative to the fiber, and cell divisions scoredas either planar or apical-basal (FIG. 8A). At 42 h after the first celldivision, Wnt7a stimulation induced planar divisions and accordinglyresulted in a 12% decrease in apical-basal cell divisions. By contrast,Vangl2 silencing increased the proportion of apical-basal cell divisionsby 15%, (n=3, ≥154 pairs, p≤1.02) (FIG. 8B). Myofibers from the sameexperiments were also stained with Pax7 and YFP antibodies, and thepercentage of symmetric cell divisions scored. A close inversecorrelation between the proportions of apical-basal versus symmetriccell divisions was observed. Wnt7a stimulation increased the proportionof symmetric cell divisions, whereas Vangl2 knock-down markedly impairedthe ability of Wnt7a to stimulate symmetric cell divisions (n=3, ≥65pairs, p≤1.02) (FIG. 8C).

To analyze the role of Vangl2 in regulating symmetric satellite cellcell divisions, fibers were cultured for 50 hours and assessed Vangl2silencing by immunostaining (FIG. 8D). At this point, Vangl2 knock-downcontinued to increase the rate of apical-basal cell divisions (n=5, ≥150pairs, p=0.001) (FIG. 8E), while depleting the population of satellitestem cells (n=3, ≥330 cells, p=0.03) (FIG. 8F). This resulted in amarked diminution in the total number of satellite cells per fibers(n=5, ≥500 cells, p=0.001) (FIG. 8G). At 3-days after knock-down ofVangl2 (FIG. 8H), a doubling in the number of cells expressing myogenin,an early marker for differentiation, was observed (n=4, ≥550 cells,p=10⁻⁵) (FIG. 8I) along with a loss of half the cells on fibers (n=4,≥550 cells, p=0.001) (FIG. 8J). Vangl2 silencing on satellitecell-derived myoblasts resulted in reduced levels of Pax7 and Myf5transcripts, along with increased levels of Myogenin (n=4, p≤1.05) (FIG.8K). Together, these data suggest that Vangl2 is required forself-renewal of both satellite stem cells and the generation oftransient-amplifying myoblasts.

These data demonstrate that Wnt7a signaling through Fzd7 requires Vangl2to induce symmetric expansion of the satellite stem cell pool. Wnt7aalso induces polarized distribution of Vangl2 protein at the oppositepoles of cells undergoing a symmetric planar cell division. Hence, Wnt7autilizes the planar cell polarity pathway to control the orientation ofsatellite cell division and their fate within the niche.

Example 5 Wnt7a Enhances Muscle Regeneration by Expanding the Stem CellPool

To investigate the role of the Wnt7a-Fzd7-Vangl2 pathway in muscleregeneration in vivo, Wnt7a was over-expressed by electroporation of aCMV-Wnt7a expression plasmid into TA muscles of 3-month old mice.Histological analysis of muscles electroporated with CMV-LacZ plasmidrevealed that majority (>80%) of the myofibers expressed theβ-galactosidase (FIG. 15A) and no regeneration deficit was detectedfollowing electroporation of control plasmid (FIG. 15B). In addition,immunostaining revealed that myofibers electroporated with CMV-Wnt7aplasmid secreted readily detectable levels of Wnt7a protein (FIG. 15C).

Notably, TA muscles electroporated with CMV-Wnt7a exhibited an 18±4%(p=0.009, n=8) increase in mass after 3 weeks. Examination of serialsections of electroporated muscles revealed an increase in the overallsize of the muscle as well as a significant increase in caliber size andnumbers of fibers throughout the body of the muscle (FIG. 9). Bycontrast, over-expression of Wnt3a resulted in a larger increase in thenumber of myofibers but these exhibited a dramatic reduction incross-sectional area, resulting in reduced regeneration efficiency (FIG.9). No effect of Wnt7a over-expression on other cell types in muscletissue was observed. However, overexpression of Wnt3a resulted inabnormal matrix deposition suggesting an enhancement of proliferation offibroblastic/smooth muscle progenitors resulting in increased fibrosis(FIG. 9B). Taken together, these results indicate that over-expressionof Wnt7a markedly enhances muscle regeneration, as evidenced by thepresence of increased numbers of larger fibers and the significantlyincreased mass of muscle.

As previously noted, Wnt7a treatment did not alter the growth ordifferentiation of activated satellite cells or primary myoblasts invitro (FIG. 14C, 16A, 16C, 16D). In addition, Wnt7a did not induce theexpression of MyoD or of Wnt/β-catenin target genes in differentiatedmyocytes (FIG. 16B, 16E). However, the in vitro experiments establishedthat Wnt7a-Fzd7-Vangl2 signaling stimulated the symmetrical expansion ofsatellite stem cells, which would then give rise to transient amplifyingprogenitors that undergo normal proliferation and differentiation. Thus,promotion of symmetric stem cell expansion with normal rates ofproliferation and differentiation of progenitor cells showed improvedtissue regeneration when compared to stimulated proliferation anddifferentiation. This is a remarkable finding and also mitigates concernof stem cell depletion.

To assess whether Wnt7a similarly stimulates the expansion of satellitestem cells in vivo, the numbers of satellite cells and satellite stemcells in regenerated muscle were assessed following electroporation ofCMV-Wnt7a. Over-expression of Wnt7a resulted in about a 2-fold increasein the number of Pax7⁺ satellite cells per myofiber on sections at 3weeks after electroporation (p=0.03, n=4). By contrast, over-expressionof Wnt3a did not alter the number of satellite cells (FIG. 10A, 10B). Toenumerate the proportion of satellite stem cells, FACS-isolatedsatellite cells were isolated from Myf5-Cre/ROSA26-YFP TA muscle at 3weeks following electroporation, cultured for 24 hours, thenimmunostained for Pax7 and YFP (FIG. 10C). Consistent with theobservations that Wnt7a induces symmetrical satellite stem celldivisions in vitro, it was observed that overexpression of Wnt7a inregenerating muscle resulted in about a 20% increase (n=5, p=0.0001) inthe proportion of Pax7⁺/YFP⁻ satellite stem cells (FIG. 10C, 10D).Therefore, these data indicate that similarly Wnt7a acts on thesatellite stem cell compartment in vitro and in vivo.

To investigate satellite stem cell function in the absence of Wnt7a, theregeneration phenotype of 3 mo-old Wnt7a^(−/−) null mice (Miller andSassoon, 1998) was examined. Quantification of Pax7-expressing satellitecells on freshly isolated myofibers from EDL muscle demonstrated thatWnt7a^(−/−) null mice exhibit about an 18% decrease in number ofsatellite cells per fiber (p=0.03, n=4) (FIG. 10E). Three-weeksfollowing a freeze-crush injury, the regenerated Wnt7a^(−/−) TA musclesappeared grossly normal (FIG. 10F), however upon closer examination, thefibers appeared to display a less uniform distribution of calibers, andthe basal lamina were of irregular thickness, consistent with a defectin regeneration. Importantly, examination of sections of regeneratedWnt7a^(−/−) TA muscles revealed a significant 36% decrease in thenumbers of satellite cells (p=0.03, n=3) (FIG. 10G). This resultstrongly supports the notion that Wnt7a plays an important role inregulating satellite stem cell function.

Overexpression of Wnt7a in muscle drives expansion of the satellite stemcell pool, and conversely, the satellite cell compartment becomesdepleted in the absence of Wnt7a. Together, these data demonstrate anovel role for Wnt7a signaling via the PCP pathway to stimulatesatellite stem cell symmetrical cell division to drive expansion.Therefore, Wnt7a regulates the homeostatic levels of the satellite stemcell compartment and thus regulates the efficiency of regenerativemyogenesis in adult skeletal muscle.

Example 6 Electroporation of Wnt7a Expression Vector in Mdx Mice Resultsin Enhanced Number of Satellite Cells and Increased Fiber Diameter

Mdx mice are a well known mouse model for Duchenne muscular dystrophy.The mdx mice harbor a mutation in exon 23 of the dystrophin generesulting in the generation of a stop codon. The mutation in thedystrophin gene leads to a disruption of the DGC complex(dystrophin-glycoprotein complex) which is crucial for the integrity ofmuscle fibers.

Referring to FIG. 17, Electroporation of Wnt7a cDNA into the TA muscleof adult wt mice leads to an increase in satellite cell numbers as wellas in the number of satellite stem cells (myf5 negative, Pax7 positive).As can be seen from the figure, the number of satellite stem cells inthe Wnt7a group was nearly doubled.

Referring to FIG. 18, the TA muscle of mdx mice was electroporated afterinjection of a control (lacZ) plasmid or a plasmid containing the codingregion of Wnt7a under control of the CMV promoter. The total number ofsatellite cells (all Pax7 positive cells) as well as satellite stemcells (myf5 negative satellite cells) was counted. The total number ofsatellite cells was significantly increased in mdx mice electroporatedwith a Wnt7a containing plasmid (p=0.005). Electroporation of a plasmidcontaining the coding region of Wnt7a lead to a significant increase inthe total number of satellite cells while the proportion of satellitestem cells to committed progenitor cells showed a tendency that moresatellite stem cells were present in mdx mice electroporated with Wnt7aplasmid.

Referring to FIG. 19, mdx mice as well as age-matched wt mice (3 monthold, male) were electroporated with either the Wnt7a containing plasmidor the lacZ control plasmid. Electroporation of Wnt7a cDNA lead to asignificant increase in fiber diameter in mdx and wt mice (p<0.001).

In summary, electroporation of Wnt7a cDNA into the TA muscle of wt aswell as mdx mice increased the number of satellite cells. Also the fiberdiameter was increased in wt and mdx mice. These findings suggest thatWnt7a could be a possible treatment for Duchenne muscular dystrophy byincreasing the number of satellite cells and thereby preventing anexhaustion of the satellite cell pool. The application of Wnt7a couldalso be used to increase healing of skeletal muscle after injury orsurgery.

Example 7 Administration of Wnt7a Recombinant Protein Produces EffectsSimilar to Electroporation of a Wnt7a Expression Vector

Referring to FIG. 20, (A) human Wnt7a protein was injected into the TAmuscle and was found to significantly enhance muscle fibre size twoweeks after injection (p<0.001) (A). The observed effects were similarto those produced by electroporation of CMV-driven mouse Wnt7a (B).

EXPERIMENTAL PROCEDURES Mice and Animal Care

Adult (8-12 weeks of age) Myf5-Cre/ROSA26-YFP mice were obtained bycrossing the knock-in Myf5-Cre (Tallquist et al., 2000) heterozygousmice with ROSA26-YFP (Srinivas et al., 2001) homozygous reporter mice.ROSA26-YFP mice were used as wild type controls. Wnt7a-null mice andtheir littermates controls were obtained by crossing heterozygousWnt7a^(+/−) mice. All mice were maintained inside a barrier facility andexperiments were performed following the University of Ottawaregulations for animal care and handling.

Cell Sorting

Mononucleated muscle derived cells were isolated from hind-limb musclesand staining was performed as previously described (Ishibashi et al.,2005; Kuang et al., 2007). Cells were separated on a MoFlo cytometer(DakoCytomation), equipped with 3 lasers. Dead cells and debris wereexcluded by Hoescht staining, and by gating on forward and side scattersprofiles (FIG. 11).

Myofiber Isolation, Culture and Immunohistochemistry

Single myofibers were isolated from the EDL muscles as previouslydescribed (Charge et al., 2002). Isolated myofibers were cultured insuspension in 6-well plates coated with horse serum to prevent fiberattachment (Kuang et al., 2006). Fibers were incubated in plating mediumconsisting of 15% FBS (Hyclone) and 1% chick embryo extract (CEE,Accurate Chemicals) in DMEM containing 2% L-glutamine, 4.5% Glucose and110 mg/ml Sodium Pyruvate. For myoblast culture, satellite cells weresorted and plated on collagen-coated dishes in Ham's F10 mediumsupplemented with 20% FBS and 5 ng/ml of basic FGF (Invitrogen). For Wntstimulation, recombinant Wnt7a or Wnt3a proteins were added in theplating medium (25 ng/ml, R&D Systems). For in vivo activation ofsatellite cells, regeneration was induced by CTX injection in the TAmuscle, and four days later, individual myofibers were isolated from theneighboring EDL muscle. Immunochemical labeling of cryosections,myofibers and cells were performed at previously reported (Kuang et al.,2006). The primary antibodies used are listed in Supplemental Table 1.

siRNA Knock-Down

For EDL myofibers, transfections were carried at 4 hours and 24 hourspost-dissection in plating medium using Lipofectamine 2000 reagent(Invitrogen) as per manufacturer's instructions. Fibers were re-fed infresh media on the next mornings and fixed after 42 hours to 72 hours ofculture. For Satellite cell-derived myoblasts, cells were re-fed 3 hoursprior to transfection and transfections were carried in growth medium.Cells were washed and re-fed with Ham's Complete media 6 hours followingtransfections. RNA was harvested 48 hours following transfection. siRNAduplexes were from Ambion siFzd7 (ID s66314), siVangl2 (ID s96802) andused at the final concentration of 10 nM each. Transfection efficiencywas monitored using Cy3-labeled siRNA. Knock-down efficiency wasassessed by real-time PCR (FIGS. 14A and 13K).

Real-Time PCR

RNA was isolated using the RNEasy kits (Qiagen) and subjected to oncolumn DNase digestion as per manufacturers instructions. cDNA synthesiswas performed using the Superscript III reverse transcriptase and randomhexamer primers (Invitrogen). Real-Time PCR was carried out aspreviously described (Ishibashi et al., 2005). Transcript levels werenormalized to GAPDH transcript levels. Relative fold change inexpression was calculated using the ΔΔCT method (CT values <30). Forrelative transcript quantification, each cDNA sample was run on a5-point standard curve as to assure a PCR efficiency of ≥95%. WntSignaling Pathway PCR Arrays were purchased from Superarray BioscienceCorporation (PAMM-043) and analysis was performed as per manufacturer′instructions. See Table 2 for primer sequences.

Statistical Analysis

A minimum of 3 and up to 5 replicates was done for experimentspresented. Data are presented as standard error of the mean. Resultswere assessed for statistical significance using Student's T Test(Microsoft Excel) and differences were considered statisticallysignificant at the p<0.05 level. Table 1 below lists antibodies used incarrying out the various experiments performed in connection with thepresent invention. Table 2 below lists PCR primers used in carrying outthe various experiments performed in connection with the presentinvention.

Muscle Injury

For freeze-induced muscle regeneration, skin and fascia of anesthetizedmice were opened and the TA muscles were subjected to three consecutivecycles of freezing-thawing by applying a liquid nitrogen-cooled metallicrod, and the wound closed by suture. For CTX-induced muscleregeneration, 25 ul of diluted cardiotoxin were directly injected intothe TA muscle without opening of the skin.

In Vivo Electroporation

40 μg of plasmid DNA in 0.9% NaCl or 0.9% NaCl (saline) was injecteddirectly into the left TA muscle of anesthetized mice, that had beenexposed by an incision through the skin. Immediately after injection,electric stimulation was applied directly to the TA by a pulse generator(ECM 830, BTX) of 100-150 volts for 6 pulses, with a fixed duration of20 ms and an interval of 200 ms using 5 mm needle electrodes (BTX).Experimental and contralateral TA muscles were isolated and embedded inOWNT7A5% Sucrose (Tissue-Tek) and frozen with isopentane cooled by coldnitrogen.

In Vivo Electroporation of MDX Mice

We electroporated mdx mice (3 month old, male, 3 animals per group) witheither 40 ug of a vector containing the coding region of the Wnt7aprotein connected to the sequence for an HA (hemagglutinin) epitopeunder control of the CMV promoter (pHANpuro-Wnt7a-HA) or thepSPORT6-lacZ vector (Invitrogen) as a control. The latter vector wasalso used to determine the efficiency of the electroporation.Electroporation was carried out as described by LeGrand et al. 2009,mice were sacrificed 2 weeks after electroporation. Electroporation wascarried out using the tibialis anterior (TA) muscle. The plasmid usedfor electroporation was dissolved in 0.9% NaCl, the total volume per TAmuscle was 40 ul. For the experiments only one TA was injected andelectroporated, the other TA was used as a control.

Sections (14 um) of the TA muscle were stained for Pax7, a marker forall satellite cells, as well as myf5, a marker for committed progenitorcells. Cells which were stained positively for both marker proteins werecounted as committed progenitor cells whereas cells only positive forPax7 were counted as satellite stem cells.

Histology and Quantification

Transverse sections (8 μm) of experimental and contralateral muscleswere cut with a cryostat (Leica CM1850). The entire TA muscles weresectioned, in order to compare experimental and contralateral muscles atthe same level on serial sections (around 400 sections were obtainedfrom each TA muscle). For LacZ reaction, cryosections were fixed with0.1% gluteraldehyde and exposed to X-gal solution. For H&E andimmonostaining, sections were fixed with 4% paraformaldehyde. Forenumeration of fibers, pictures of laminin-stained cryosections wereassembled and counted on Adobe Photoshop CS2. Quantification ofmyofibers caliber was performed with ImageJ. The satellite cellenumeration was performed on Photoshop, on pictures of Pax7 and Lamininco-immunostained cryosections taken in regenerated areas where all thefibers had centrally located nuclei. “Satellite Cell Content” representsthe number of sub-laminar Pax7+ve satellite cells normalized per fibernumber, and to the contralateral leg.

Subtractive Hybridization

RNA samples (10 ng) from sorted YFP+ and YFP− satellite cells wereamplified using the Super-SMART cDNA amplification kit (Clontech)following the protocols provided. 2 ug of representative cDNA from eachsample were used to generate a subtractive library with the PCR-Selectkit (Clontech) as per manufacturer′ instructions. The amplified cDNAfrom sorted YFP⁻ was used as “tester” and the amplified cDNA from sortedYFP⁺ was used as “driver”. SSH products were cloned in pCR®2.1 vectorusing a TA cloning Kit (Invitrogen) and 200 clones from the SSH librarywere sequenced with nested primers using the ABI 3730 DNA Analyzers(Applied Biosystems), according to the manufacturers' protocols andmanuals.

TABLE 1 Antibodies Catalog Dilution Supplier number α7-Integrin 1/200MBL International K0046-3 Active-β-Catenin 1/500 Millipore Corp 05-665CD11b 1/200 eBiosciences 12-0112 CD144 1/200 BD Biosciences 555289 CD311/200 eBiosciences 12-0451 CD34 1/50 BD Biosciences 553732 CD45 1/200eBiosciences 12-0311 Celsr2 1/200 MBL International LS-A1943 CXCR4 1/100BD Biosciences 551968 Frizzled7 1/100 R&D Systems MAB-1981 GFP 1/500Invitrogen A21311 Laminin α2 1/200 Alexis Biochemicals ALX-804-190 Myf51/50 Santa Cruz Biotechnology sc-302 Inc MyoD 1/50 Santa CruzBiotechnology sc-304 Inc Myogenin 1/100 Santa Cruz Biotechnology sc-578Inc Myosin Heavy 1/20 DSHB MF20 Chain Pax7 1/10 DSHB PAX7 Prickle1 1/100Abcam Ab15577 Sca1 1/202 eBiosciences 12-5981 Syndecan4 1/5000 Gift fromBrad Olwin Vangl2 1/200 Santa Cruz Biotechnology sc-46561 Inc Wnt7a1/100 Santa Cruz Biotechnology sc-26360 Inc

TABLE 2 PCR Primers Primer Sequence Exon Amplicon SEQ ID NO Axin2_FAAGAGAAGCGACCCAGTCAA  2 198 13 Axin2_R CTGCGATGCATCTCTCTCTG  3 14Celsr1_F GGGGACTACTGCGAGACTGA  2 177 15 Celsr1_R CCCGTTTTTGCATACTCCAC  316 Celsr2_F CCGAGGTGGACCTCTGTTAC  2 186 17 Celsr2_R CCACCAACAGGTTGACACAG 3 18 Celsr3_F ATGACCCGGATGTCTCTGAC  1 216 19 Celsr3_RACTCCTCCGTGATGATGACC  2 20 Dvl1_F CTTACCAGGACCCTGGCTTC 11 246 21 Dvl1_RCCTGACTTCGAGGGCTACTG 12 22 Dvl2_F TATGTCTTCGGGGACCTCAG 13 212 23 Dvl2_RCGAAGAAAGCTCGTGGTAGG 14 24 Dvl3_F GCCTATGGCTTTCCCTTACC 14 198 25 Dvl3_RACTTGGAGTCCCCAGCTTTT 15 26 Fzd1_F CAAGGTTTACGGGCTCATGT  1 180 27 Fzd1_RGTAACAGCCGGACAGGAAAA  1 28 Fzd3_F CCTTGAGGATGTGCCAAGAT  2 178 29 Fzd3_RGCTATAGGCACGCTGACACA  3 30 Fzd4_F AACCTCGGCTACAACGTGAC  1 150 31 Fzd4_RTGGCACATAAACCGAACAAA  2 32 Fzd6_F AATGGACACTTTTGGCATCC  3 223 33 Fzd6_RAGGGGCACACTGTTCAATTC  4 34 Fzd7_F GCTTCCTAGGTGAGCGTGAC  1 216 35 Fzd7_RAACCCGACAGGAAGATGATG  1 36 Gapdh_F ATGCCAGTGAGCTTCCCGTC  1 470 37Gapdh_R CATCACCATCTTCCAGGAGC  1 38 Myf5_F TGAAGGATGGACATGACGGACG  1 13339 Myf5_R TTGTGTGCTCCGAAGGCTGCTA  1 40 MyoD_F TACCCAAGGTGGAGATCCTG  1200 41 MyoD_R CATCATGCCATCAGAGCAGT  1 42 Myogenin_F GAAAGTGAATGAGGCCTTCG 1 248 43 Myogenin_R ACGATGGACGTAAGGGAGTG  3 44 Notch3_FGATGACACATCAGCCAGCAT 32 248 45 Notch3_R GAGCGGTTCCTGATGAGAAT 33 46Pax7_F CTGGATGAGGGCTCAGATGT  3 245 47 Pax7_R GGTTAGCTCCTGCCTGCTTA  4 48Tcf7_F CCCCAGCTTTCTCCACTCTA  4 165 49 Tcf7_R TCACAGTATGGGGGAGCTGT  5 50Vangl1_F GCACTACCACAGCATGGAGA  7 235 51 Vangl1_R ATTGACCACGAGGCTGAAGT  852 Vangl2_F CCCCAGTTCACACTCAAGGT  4 157 53 Vangl2_R ACTTGGGCAGGTTGAGGAG 5 54 GCACGACTTCTTCAAGTCCGCCATG 55 Yfp_F CC  1 263GCGGATCTTGAAGTTCACCTTGATG 56 Yfp_R CC  1

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The above-described embodiments of the invention are intended to beexamples only. Alterations, modifications and variations can be effectedto the particular embodiments by those of skill in the art withoutdeparting from the scope of the invention, which is defined solely bythe claims appended hereto.

The invention claimed is:
 1. A method for modulating division symmetryof satellite stem cells comprising contacting isolated satellite stemcells with a composition comprising as an active agent a Wnt7apolypeptide or an active variant thereof having at least 95% identity toSEQ ID NO:2 or SEQ ID NO:4 and capable of binding to and activatingFzd7.
 2. A method for increasing the number of satellite stem cells in askeletal muscle in a subject in need thereof comprising contactingisolated satellite stem cells with a composition comprising as an activeagent a Wnt7a polypeptide or an active variant thereof having at least95% identity to SEQ ID NO:2 or SEQ ID NO:4 and capable of binding to andactivating Fzd7, and administering the satellite stem cells to theskeletal muscle.
 3. The method of claim 2, wherein the subject has adegenerative disease.
 4. The method of claim 3, wherein the degenerativedisease is a muscular dystrophy.
 5. The method of claim 4, wherein themuscle dystrophy is muscular dystrophy selected from the groupconsisting of Duchenne muscular dystrophy (DMD), Becker musculardystrophy (BMD), Emery-Dreifuss muscular dystrophy, Landouzy-Dejerinemuscular dystrophy, facioscapulohumeral muscular dystrophy (FSH),Limb-Girdle muscular dystrophies, von Graefe-Fuchs muscular dystrophy,oculopharyngeal muscular dystrophy (OPMD), Myotonic dystrophy(Steinert's disease) and congenital muscular dystrophies.
 6. The methodof claim 2, wherein the subject suffers from muscle wasting or atrophyassociated with injury or illness.
 7. A method for expanding apopulation of satellite stem cells comprising isolating satellite stemcells and contacting the isolated satellite stem cells with an effectiveamount of a composition comprising a Wnt7a polypeptide or an activevariant thereof having at least 95% identity to SEQ ID NO:2 or SEQ IDNO:4 and capable of binding to and activating Fzd7.